Berk2010-Conor: Difference between revisions

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[[Berk2010-Conor (June) | June Entries]]
[[Berk2010-Conor (June) | June Entries]]<br>
==To Do:==
[[Berk2010-Conor (July) | July Entries]]
*Choano Assays
*next stage of 2AB assembly for parts 007 and 013


==[[User:Conor McClune|Conor McClune]] 13:52, 26 July 2010 (EDT)==
===Assembly of iGEM10_012===
===='''Picking Colonies'''====
*Picked 5 colonies
*Swiped on AKC plate to check for cotransformation
*dipped in Col PCR MM containing the oligos ca998 and igemTen18
**expected size: 1376bp


===Eco/Bam Transfer of iGEM10_006 (2nd attempt)===
==To Do:==
====Ligation====
*used the Eco/Bam version of iGEM10_006 that I digested and gel purified yesterday
*recipe: 5uL water, 1uL ligase buffer, 1uL ligase, 1uL digested pMLL6-KA vector, 2uL Eco/Bam digested part
*incubated at room temp from 8:15-8-45pm


====Transformation====
==[[User:Conor McClune|Conor McClune]] 22:47, 16 October 2010 (EDT)==
*Cell mixture: 100uL JTK030 cells  + 20uL KCN
===Transposase Assembly===
* I added 80uL of this mixture to my ligation mixture
====Colony PCR analysis====
*let sit on ice for 5 minutes
Colony PCR failed: no bands
*heat shocked for 90 seconds
*let sit on ice for 1min
*added 100uL 2YT
*rescued from 8:55-


==[[User:Conor McClune|Conor McClune]] 15:47, 23 July 2010 (EDT)==
*171+174 (A-D):
===Restriction mapping of iGEM10_006 Eco/Bam===
**lanes 1-4
*digested all 5 minipreps with Eco/Bam
**expected size: 4615
*4uL DNA, 4uL H20, 1uL each enzyme, 1uL NEB2
**good clones:
*incubated 11:15-12:12
*171+175 (A-B):
[[image:ConorGel65.jpg|400px]]
**lanes 5-6
**expected size: 4416
**good clones:
*170+175 (A-C):
**lanes 7-9
**expected size: 5265
**good clones:
*170+174 (A-B):
**lanes 10-11
**expected size: 5464
**good clones:
*170+173 (A-D):
**lanes 12-15
**expected size: 5471
**good clones:


===Assembly of iGEM10_012===
==[[User:Conor McClune|Conor McClune]] 18:11, 13 October 2010 (EDT)==
'''Digestion'''<br>
Digested iGEM10_010 with lefty MM and iGEM10_011 with righty MM
*incubated 11:25-12:25
2:00-
*expected sizes:
**010:
***2912, 1064
**011:
***4834, 1475


'''Gel Purification'''<br>
===Transposase assembly===
[[image:ConorGel66.jpg|100px]]<br>
Conducted the following digestions:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Restr. Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Parent Size'''
| align="center" style="background:#f0f0f0;"|'''Expected Fragments'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
| align="center" style="background:#f0f0f0;"|'''Gel Purified'''
|-
| 1||163-5a||Xho/Bam||6004||4730, 1270||yes||
|-
| 2||163-5b||Xho/Bam||6004||4730, 1270||yes||yes
|-
| 3||163-10a||Xho/Bam||5150||3880, 1270||no||
|-
| 4||163-10b||Xho/Bam||5150||3880, 1270||no||
|-
| 5||163-17b||Xho/Bam||5400||4100, 1270||yes||yes
|-
| 6||163-17a||Xho/Bam||5400||4100, 1270||yes||
|-
| 7||ladder||||||||||
|-
| 8||163-17b||Xho/Bgl||5400||1475, 3921||yes||
|-
| 9||2142-164a||Xho/Bgl||5090||3408, 1680||yes||yes
|-
| 10||2142-164b||Xho/Bgl||5090||3408, 1680||yes||
|-
| 11||2142-165a||Xho/Bgl||5080||3401, 1680||yes||yes
|-
| 12||2142-165b||Xho/Bgl||5080||3401, 1680||yes||
|-
| 13||2142-166a||Xho/Bgl||4880||3200, 1680||yes||
|-
| 14||2142-166b||Xho/Bgl||4880||3200, 1680||yes||yes
|}
Gel image:<br>
[[image:ConorGel105.jpg|400px]]


'''Ligation'''<br>
===preparation for plasmid payload cytometry===
I did a standard ligation recipe and let it sit from 4:00-4:30
====Self lysis test====
<br>
*started 2:00
'''Transformation'''<br>
*checked at 4:30:
*Cell mixture: 100ul JTK030 cells with 20ul KCN
{| {{table}}
*Combined 70uL ligation mixture with ligation mixture
| ||1||2||3||6||7
*heat shocked
|-
*rescued for 1.5 hours
| 51||lysed||lysed||lysed||barely lysed||lysed
*plated entire solution onto KA plate
|-
| 51||lysed||lysed||lysed||lysed||lysed
|}


===Repicking Colonies of iGEM10_006 Eco/Bam Transfer===
I may have destroyed my DNA samples by vortexing during the wrong stage of miniprepping, so I am going to grow them up again. There are only 5 colonies, and we know from the last colony PCR that they are all on the correct vector. This time I shall run a Col PCR that will check the part.
*Repicking all 5 colonies
*running col PCR with oligos: ca998 and igemTen004
''Gel Results''<br>
Expected size: 1859 <br>
[[image:ConorGel68.jpg|400px]]<br>
These bands are all too short. I will have to redigest and retransform.


===Gel Analysis of iGEM10_006 Eco/Bam digest===
====preparation of cells====
Expected size: 5925 <br>
*scraped choanos and put 1ml in each test tube
[[image:ConorGel67.jpg|400px]]<br>
*added 40 uL bacteria to each test tube at 2:30
There are no visible bands. This may have been a faulty digestion and could possibly be the source of the error. I will redigest it
*induced with arabinose fifteen minutes later (at 2:45)


===Re-Eco/Bam Transfer of iGEM10_006 to KA===
==[[User:Conor McClune|Conor McClune]] 18:31, 12 October 2010 (EDT)==
*Did a standard Eco/Bam digestion recipe for iGEM10_006.
*flow cytometry got no hits, because the choanos were in ASW, so the bacteria did not lyse
*Incubated from 4:06-4:45. Gel purified the larger band.


==[[User:Conor McClune|Conor McClune]] 17:24, 22 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 18:31, 11 October 2010 (EDT)==
===Picking Colonies/ Colony PCR for iGEM10_007===
===preparation for plasmid payload cytometry===
*Picking 24 colonies
====Self lysis test====
*colony PCR's:
*started 2:15
**ca56 + igemTen004
*checked at 4:15:
***checks for iGEM10_001 and sbb04 (left side of iGEM10_007)
{| {{table}}
***expected size: 2045
| ||1||2||3||6||7
 
|-
''Gel Results''<br>
| 51||lysed||lysed||lysed||lysed||lysed
[[image: ConorGel64.jpg|400px]]
|-
*all lanes are empty
| 51||lysed||lysed||lysed||lysed||lysed
 
|}
==[[User:Conor McClune|Conor McClune]] 13:52, 21 July 2010 (EDT)==
===NH4 Toxicity Assay===
====Setup (completed 9:15am)====
*made 4 aliquots of 3mL of healthy MbFb (2 days after 1:15 split)
*placed these in the following conditions:
**25*C
**37*C
**37*C with 10mM NH4
**37*C with 1mM NH4
 
====1 Hour Count (10:15am)====
*the PI stain seems to be dead, because I can see no fluorescence, including background. Thus I will only take pictures and qualitative observations
*increased swimming in all three 37*C samples, otherwise, no difference between samples
 
====4 Hour Observations (1:15 pm)====
*choanos are still much more active at 37*C than at 25*C
 
====12th Hour (9:15pm)====
Choanos are still active in all wells. I took a hemocytometer count of the cells in each wells. However, I did not dilute the first two cultures (which were denser than I expected) and counting took longer than 30 minutes (the usual time before formaldehyde begins causing choanos to lyse). Thus, the counts for the last two cultures may be inaccurate.


Choano Counting Procedure:
*scrape choanos
*remove 200uL of each of the 4 cultures to 1.5mL tubes
*add 1uL formaldehyde to each tube, shake
*pipette 10uL of each tube into hemocytometer
*wait 10 minutes for choanos to sink
*count 4 quadrants and average:


Note: due to time crunch, I diluted the two NH4 samples 1:10 in order to speed up the process
====preparation of cells====
*scraped choanos and put 6ml in each test tube
*added 240 uL bacteria to each test tube
*induced with arabinose fifteen minutes later (at 2:30)


==[[User:Conor McClune|Conor McClune]] 19:02, 6 October 2010 (EDT)==
===Plasmid Delivery Assay===
''Plasmid Payload Numbers:''
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Number'''
| align="center" style="background:#f0f0f0;"|'''Dilution'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''Antibiotic'''
| align="center" style="background:#f0f0f0;"|'''2'''
|-
| align="center" style="background:#f0f0f0;"|'''3'''
| 1||pDYAL eGFP||Amp
| align="center" style="background:#f0f0f0;"|'''4'''
|-
| align="center" style="background:#f0f0f0;"|'''Average'''
| 2||pDYAL eGFP Swar||Amp
| align="center" style="background:#f0f0f0;"|'''Cell density (cells/mL)'''
|-
| 3||Clipit GFP||Amp
|-
| 4||M cherry||Amp
|-
|-
| 25*C control||n/a||242||309||249||270||267.5||2675000
| 5||Tera\'s α-tub GFP jtk159||Amp
|-
|-
| 37*C control||n/a||284||321||290||274||292.25||2922500
| 6||Tera\'s efla GFP jtk160||Amp
|-
|-
| 37*C 10mM NH4||1 :10||27||31||29||6||23.25||2325000
| 7||PfuGWP GFP||Amp
|-
|-
| 37*C 1mM NH4||1 :10||17||30||17||24||22||2200000
| 8||Tera\'s myo GFP jtk162||Amp
|}
|}


At 9:30, I had scraped the cells to do the above counts, so by 10:30 the percentage of choanos attached the the bottom had peaked. I aspirated all the media from each of the 4 cultures and replaced it with 3mL choano media.
*51 and 52 refer to the Self-lysis/Vacuole-buster devices iGEM10_051-052
 
====25th Hour====
I removed 100uL of each culture, diluted it 1:4 and counted it on the hemocytometer. This should be a more accurate count than the one at 12 hours.:


===Procedure===
''Choanos''
*aspirated two plates (15ml each) of choanos in ASW, resuspended each in 5mL of choano media
**note: the choano media we planned on using was contaminated, so I had to use an older one (one that seemed to be lacking something because choanos have not grown very well in it)
*scraped choanos, mixed the two plates (to get 10mL), aliquoted 500uL into each of 12 mini slide wells
*added 20uL of bacteria to the wells (10:15am):
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| 1+51||2+51||3+51||6+51||7+51||lifeact iGEM10_171 +51(color control)
| align="center" style="background:#f0f0f0;"|'''Dilution'''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''3'''
| align="center" style="background:#f0f0f0;"|'''4'''
| align="center" style="background:#f0f0f0;"|'''Average'''
| align="center" style="background:#f0f0f0;"|'''Cell density (cells/mL)'''
|-
| 25*C control||1 :4||31||29||31||25||29||1160000
|-
| 37*C control||1 :4||13||16||16||16||15.25||610000
|-
| 37*C 10mM NH4||1 :4||14||11||10||7||10.5||420000
|-
|-
| 37*C 1mM NH4||1 :4||16||15||12||11||13.5||540000
| 1+52||2+52||3+52||6+52||7+52||white bacteria
|}
|}
''Results:'' The choanos clearly recovered from the NH4. At this point it looks like the temperature difference is more detrimental to growth than NH4.
*mistake:added 0.5uL ATC to each well at 10:30am, instead of arabinose (SLD is under Pbad not Ptet)
*correction: added 5uL arabinose to each well at 11:00am
 
''Self lysis test''
**made two 2mL aliquots of each of the plasmid bacteria cultures listed above
**added 2uL ATC (mistake) and 20uL of arabinose to one of each of the aliquots of each culture
 
===Results===
''Self Lysis Test''
*after 5 hours, all 10 plasmid+SLD/VB cultures with arabinose had lysed.
 
==[[User:Conor McClune|Conor McClune]] 22:30, 26 September 2010 (EDT)==
===Eco/Bam transfer of iGEM10_171 (A) into Bca1256===
*transformed product into JTK159  and plated on spec
===Re-Assembly of iGEM10_170===
*gel purified my previous digestions of iGEM10_159 and Bjh1906
*ligated 4uL of each with 1uL t4 buffer and 1uL t4 ligase
*transformed into JTK159 and plated on CA


===Picked colonies for iGEM10_011===
==[[User:Conor McClune|Conor McClune]] 16:34, 23 September 2010 (EDT)==
Picked 8 colonies and ran a Col PCR with standard Col PCR MM (caa998 + G00101)
===LifeAct Assembly===
Of the colonies if iGEM10_170 and iGEM10_171 picked yesterday, colonies 5,8,12 and 15 were cotransformed


''PCR Gel Results:''<br>
Miniprepped: Colonies 1,2,3,7 (iGEM10_170) and colonies 10,11,18, 20 (iGEM10_171) because they were the reddest
Expected size: 3700bp <br>
*renamed these clones iGEM10_170 A-D and iGEM10_171 A-D, respectively
[[image:ConorGel63.jpg|400px]]
**iGEM10_171A sequenced well, with one mutation in the promoter
*lanes 3-8 look perfect
**all of iGEM10_170 A-D are missing the terminator
*Colonies 1-2 are cotransformed.


To miniprep: iGEM10_011 #3,4
==[[User:Conor McClune|Conor McClune]] 19:55, 22 September 2010 (EDT)==
===LifeAct Assembly===
There is growth on the plates I made yesterday for iGEM10_170-171, though the Red/green colonies on iGEM10_171 are very small


===Miniprepped iGEM10_006 #1-5===
I picked 8 colonies for iGEM10_170 and 12 from iGEM10_171 (only the last 4 may be green AND red).  
Note: Vortexed briefly after adding P2, which may cause some contamination with genomic DNA.


===Assembly of iGEM10_007===
Ran a colony PCR, but it did not show much because the addition of the terminator is not visible on the gel. The gel for the last colonies
'''Digestion'''<br>
*iGEM10_001 a - did standard Xho/Bam digestion
*iGEM10_006 #1-5:
**digested all 5 minipreps; this digestion will serve as both analytical and practical.
**made a master mix: 20uL H20, 5uL NEB2, 5uL each of Bgl and Xho
**mixed 7uL of mastermix with 4uL of each miniprep
I decided to gel purify the gels, since I was running a gel. Expected sizes:
*iGEM10_006: 6994, 1608
*iGEM10_001: 2810, 1196
[[image:ConorGel62.jpg|400px]]


==[[User:Conor McClune|Conor McClune]] 20:08, 20 July 2010 (EDT)==
[[image:ConorGel104.jpg|200px]]
===Reassembly of iGEM10_011===
*lanes 1-8: iGEM10_170
iGEM10_011 once again did not grow, so I suspect my digestion of iGEM10_008 may be bad and should be redone.
**all but colony 8 look good
*lanes 9-13: iGEM10_171
**all look good, except that col PCR failed for colony 12


'''Digestion'''<br>
==[[User:Conor McClune|Conor McClune]] 18:18, 21 September 2010 (EDT)==
Did a standard Xho/Bam digestion of iGEM10_008d and gel purified the larger part (2.5kb)''
===Restriction digest (Eco/Bam)of iGEM10_159-160===
*used iGEM10_159C
*expected band sizes: 2660, 1580
[[image:ConorGel103.jpg|200px]]
*the bands look good for both (faintess can be attributed to a bad zymo-forgot to spin out solvent for 90 sec before adding water)


===Assembly of iGEM===
Digested:
*Xho/Bam:  iGEM10_159C and iGEM10_160
*Xho/Bgl: Bjh1906<br>
Ligated each of the iGEM parts to left of Bjh1906 to make iGEM10_170-171, respectively
<br>
<br>
'''Ligation'''<br>
Transformed into JTK159 cells and plated on CA
Ligated my gel purified fragment with Amy's gel purified Xho/Bgl digestion of Bjh2294.<br>
 
==[[User:Conor McClune|Conor McClune]] 19:59, 18 September 2010 (EDT)==
===LifeAct Assembly===
====Redigestion of iGEM10_156A and iGEM10_159D====
(this is necessary because the previous version of
*Both were digested with Xho/Bam for 30 min
*Zymo'd
*Ligation (5:00-5:30):
**iGEM10_156A + Bjh1881
**iGEM10_159D + Bjh1906 (terminator)
**iGEM10_159A + Bjh1906 (terminator)
 
''Results:'' no growth for the iGEM10_159 + Bjh1906 assemblies, but one red/green colony for iGEM10_156A + Bjh1881 = iGEM10_160
*colony PCR of iGEM10_160:
[[image:ConorGel102.jpg|100px]]
*expected band : 1800bp, the band looks good


====Remaking iGEM10_161 and iGEM10_162====
(This is necessary because I had mislabelled iGEM10_157-158)<br>
yesterday:
*ligated the predigested combinations:
**iGEM10_157 + Bjh1881
**iGEM10_158 + Jtk2541
today:
*transformed both ligations into JTK159 strain
*plated on CK


'''Transformation'''<br>
''Results:'' no growth, failed assembly
Cell mixture: 100uL jtk030 cells, 30uL kcm, 50uL h20.
I added 90uL of this mixture to my ligation mixture, incubated for 1hr and plated the entire solution on a CA plate.


===Picking Colonies for iGEM10_006 Eco/Bam Transfer into KA===
==[[User:Conor McClune|Conor McClune]] 18:38, 17 September 2010 (EDT)==
Only five colonies grew, so I picked all 5. I swiped each colony on an AKC plate to check for cotransformation, and dipped it in a colony PCR mix containing the oligos igemTen10 and TH313.
===LifeAct Assembly===
====Restriction digest of iGEM10_159-162  with Eco/Bam====
* expected sizes for all bands: 2660, 1580
*iGEM10_159: lanes 1-4
*iGEM10_160: lanes 5-8
*iGEM10_161: lanes 9-12
*iGEM10_162: lanes 13-16
[[Image:Conor 9-14-10.JPG|400px]]


''PCR Gel Results''<br>
[[Image:Conor 9-14-10 2.JPG|400px]]
Expected size: 1808 <br>
Incorrect Size: 800 <br>
[[image:ConorGel61.jpg|400px]]
*all five parts appear to be in the correct vector.
*None of the colonies were cotransformed


===Miniprep of iGEM===
==[[User:Conor McClune|Conor McClune]] 18:38, 14 September 2010 (EDT)==
I miniprepped iGEM10_004 #8 and iGEM10_010 #1-3.<br>
===Assembly of LifeAct Payload===
note:I accidentally used Zymo tubes (which decreases yield), so I only eluted with 25uL H20
'''Potential Error discovered:''' ''plate with Eco/Bam iGEM157 and iGEM10158 were probably mislabelled since 157 was red and 158 was green (they should have been the green and red, respectively). This means that iGEM161-162 are probably just two copies of the same FP, one lifeact and one not.''


===Restriction Mapping of iGEM10_004,iGEM10_005, iGEM10_010===
==[[User:Conor McClune|Conor McClune]] 22:03, 12 September 2010 (EDT)==
==== Yesterday's Minipreps ====
===Lifeact Payload assembly===
[[image:ConorGel59.jpg|400px]]
====Digestion====
{| {{table}}
*Lefty digested: iGEM10_155-158
| align="center" style="background:#f0f0f0;"|'''Lane'''
*Righty digested: Bjh1881, Jtk2541
| align="center" style="background:#f0f0f0;"|'''Part'''
*incubated 3:30-4:20
| align="center" style="background:#f0f0f0;"|'''Colony #'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Expected Sizes'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_004||8||pMLL9-CA||Eco/Bam||3580, 2736||probably
|-
| 2||igEM10_005||1||pMLL8-KC||Eco/Bam||2662, 2163||yes
|-
| 3||igEM10_005||5||pMLL8-KC||Eco/Bam||2662, 2163||yes
|-
| 4||igEM10_005||6||pMLL8-KC||Eco/Bam||2662, 2163||yes
|-
| 5||igEM10_005||8||pMLL8-KC||Eco/Bam||2662, 2163||yes
|}


====Today's Minipreps====
====Ligation====
[[image:ConorGel60.jpg|400px]]
*ligating the following combinations:
{| {{table}}
**iGEM10_155 + Jtk2541
| align="center" style="background:#f0f0f0;"|'''Lane'''
**iGEM10_156 + Bjh1881
| align="center" style="background:#f0f0f0;"|'''Part'''
**iGEM10_157 + Bjh1881
| align="center" style="background:#f0f0f0;"|'''Colony #'''
**iGEM10_158 + Jtk2541
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Expected Sizes'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_004||8||pMLL9-CA||Eco/Bam||3580, 2736||probably
|-
| 2||igEM10_010||1||pMLL8-KC||Eco/Bam||2662, 1314||yes
|-
| 3||igEM10_010||2||pMLL8-KC||Eco/Bam||2662, 1314||yes
|-
| 4||igEM10_010||3||pMLL8-KC||Eco/Bam||2662, 1314||yes
|}


==[[User:Conor McClune|Conor McClune]] 15:37, 19 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 01:09, 10 September 2010 (EDT)==
===Sequence Confirmation of iGEM10_006 #3===
===Lifact Assembly===
I sequenced iGEM10_006 #3 with the reverse oligo igemTen008. This part does not quite reach the junction I was trying to make. I will try sequencing it again using igemTen003.
*Eco/Bam'd iGEM10_155-158 into pMLL9 (CA) (for continued assembly) and also into 1256 (Spec) (for assaying in choanos)


===Eco/Bam Transfer of iGEM10_006 #3===
==[[User:Conor McClune|Conor McClune]] 01:28, 9 September 2010 (EDT)==
'''Digestion'''<br> 4uL H20, 4uL DNA, 1uL Eco, 1uL Bam, 1uL NEB2. Digested from 11:05-12:10 <br> I also needed to digest more pMLL6-KA, since our stock is empty. to do this, I mixed 10uL miniprepped DNA (containing pMLLKA) with 10uL H20, 2.5uL NEB2, 1.25uL Eco, 1.25 Bam. Digested from 1:49-2:23
===Lifact Payload Assembly===
*I Eco/Bam digested iGEM10_155-158
*Gel purified smaller fragment of each
*meant to eco/bam into pMLL CA but we were out of the digested vector. I will have to digest this tomorrow


<br>
==[[User:Conor McClune|Conor McClune]] 18:04, 7 September 2010 (EDT)==
'''Gel Purification'''<br>
===Lifact Assembly===
Expected bands:
====Colony PCR results====
*iGEM10_006
*colony # corresponds to lane #
**desired - 5700
*expected sizes:
**undesired - 2800
**lanes 1-4 Bca1237 + Bjh2252:1070
*pMLL6-KA
**lanes 5-8 Bca1238 + Bjh2252:1070
**desired - 2877
**lanes 9-4 Bca1237 + Bjh2251:1020
**undesired - 324
**lanes 1-4 Bca1238 + Bjh2251:1020
Zymo purified the desired products. Eluted iGEM10_006 with 8.5 uL water, and pMLL6-KA with 20uL water


'''Ligation'''<br>
[[image:ConorGel101.jpg|400px]]
Standard ligation procedure and recipe.
*note: broken well in lane 8, so contents were put in adjacent well
Results:
*all lanes look good


===Assembly of iGEM10_011===
==[[User:Conor McClune|Conor McClune]] 21:06, 5 September 2010 (EDT)==
No colonies grew up for iGEM10_011, so I have to re-assemble it. I already have a gel purified
===Lifact Payload Assembly===
*Ran a gel of Tahoura's PCR of Pcon parts Bca1237 and Bca1238:
**image: [[image:ConorGel100.jpg|100px]]<br>
**cut out 200-300bp range for both parts and small fragment gel purified (eluted with 8.5uL H20)
*added 1uL each of NEB2, Bam and Eco to each of the purified products:
**incubated at 37*C from 5:50-


===Picking Colonies for iGEM10_004 and iGEM10_010===
==[[User:Conor McClune|Conor McClune]] 21:15, 13 August 2010 (EDT)==
*No colonies grew up for iGEM10_011.
===Assembly of iGEM10_146 and iGEM10_149===
*8 Colonies picked from each plate.
====PCR magnification of Pcon part Bca1152====
*iGEM10_010 colonies dipped in a colony PCR MM with oligos ca998 and G00101
*template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca9145
*iGEM10_004 colonies dipped in two Col PCR MM:
*recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
**ca998 and G00101
*run on 2K55
**igemTen10 and TH313
<br>
<br> ''PCR Gel Results''<br>
'''Gel Verification'''
[[image:ConorGel58.jpg|400px]]
*expected size:206
*cotransformation plate reveals that iGEM10_004 colonies #1 and #4 are cotransformed
*gel purified the
{| {{table}}
The Expand seems to have worked best. It has a strong band at 200bp
| align="center" style="background:#f0f0f0;"|'''Lanes'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable Lanes'''
|-
| 1 to 8||iGEM10_004||pMLL9-CA||ca998||G00101||3771||8 (possibly)
|-
| 9 to 16||iGEM10_004||pMLL9-CA||igemTen10||TH313||1808||9,12,16
|-
| 17 to 24||iGEM10_010||pMLL8-KC||ca998||G00101||1505||17-19,21-24
|}
to miniprep: iGEM10_004 #8, iGEM10_010 #1-3


==[[User:Conor McClune|Conor McClune]] 16:00, 16 July 2010 (EDT)==
====Digestions====
===Assembly of iGEM10_010-11===
*for Bca1152 (Pcon)-recipe:
'''Digestion:'''<br>
**8uL of eluted PCR product
iGEM10_009 and iGEM10_008 are already digested and gel purified<br>
**1uL of NEB Buffer 2
*used methylated 2:1 diluted minipreps of Bjh2294 and sbb10
**1uL EcoRI
*recipe:8uL DNA, 4uL water, 1uL NEB2, 1uL each enzyme
**1uL BamHI
*digested sbb10 with Xho/Bam, digested Bjh2294 with Xho/Bgl
*for Bjh2252(lifeact gfp) and Bjh2251 (lifeact rfp):
*digested from 12:50-1:40 <br>
** 14uL ddH2O
** 2 uL NEB2+ATP Buffer
** 1uL EcoRI
** 1uL BamHI
** 1uL miniprepped plasmid
*incubated for 1hr at 37
*zymo cleaned FP digests (eluted with 20uL), small fragment zymo cleaned Bca1152 (
====Ligation====
*recipe:
**10uL vector digest (Bjh2251 or Bjh2251)
**7.5uL Bca1152
**2uL Ligase
**2uL T4 buffer +ATP
*let sit on counter for 30 min


''Ligation''
====Transformation====
*Recipe: 6.5uL H20, 1uL each DNA, 1uL T4 Buffer, 1uL Ligase
*cell mixture: 100uL Jtk030, 15uL KCM
*I ligated the following combinations using the above digestions and stored digestions from my freezer box:
*50uL of cell mixture added to each ligation mixture
{| {{table}}
*let sit on ice for a few minutes
| align="center" style="background:#f0f0f0;"|'''Part 1'''
*heat shocked at 42*C for 90 sec
| align="center" style="background:#f0f0f0;"|'''Part 2'''
*added 100uL 2yt
| align="center" style="background:#f0f0f0;"|'''Product'''
*rescued for 45 min
|-
*plated both on CA
| sbb10||iGEM10_009||iGEM10_010
|-
| iGEM10_008||Bjh2294||iGEM10_011
|-
| iGEM10_004||pMLL9-CA||iGEM10_004
|}


==[[User:Conor McClune|Conor McClune]] 14:40, 12 August 2010 (EDT)==
===PCR magnification of Pcon part Bca1152===
*template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
*recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
*run on 2K55
<br>
<br>
'''Transformation'''
'''Gel Verification'''
*started with 100ul comp jtk030 cells
*expected size:1079
*added 100ul H20 and 40uL KCN
 
*added 70uL of cell mixture to each ligation
[[image:ConorGel98.jpg|100px]]<br>
*conducted standard heat shock transformation with 1hr incubation
The band looks too long
*plated iGEM10_010 on CK and iGEM10_011/iGEM10_004 on CA
 
===Analytical PCR of iGEM10_006 #3===
===Assembly of iGEM10_146 and iGEM10_149===
Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
====Digestion====
{| {{table}}
*standard Eco/Bgl digestion of Bjh2252 (GFP) and Bjh2251 (RFP)
| align="center" style="background:#f0f0f0;"|'''Well'''
*11:50-1:00
| align="center" style="background:#f0f0f0;"|'''Part'''
*product was zymo purified
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_006||ca998||igemTen004||1859||yes
|-
| 2||iGEM10_006||ca998||igemTen006||2225||yes
|-
| 3||iGEM10_006||ca998||igemTen008||3289||yes
|}


[[image:ConorGel57.jpg|400px]]
====Ligation====
*recipe: 3uL H20, 1uL T4 buffer, 1uL T4 ligase, 1uL FP part (Bjh2252 or Bjh2251), 4uL yesterdays Eco/Bam digest of Expand PCR of Bca1152
*incubated on desk from 11:50-12:30


==[[User:Conor McClune|Conor McClune]] 15:43, 15 July 2010 (EDT)==
====Transformation====
===Picking Colonies/Colony PCR for Eco/Bam transfers (sbb18, iGEM10_004-5)===
*cell mixture: 200uL JTK030 cells, 30uL KCM
*picked 8 colonies of sbb18, iGEM10_004, iGEM10_005
*added 70uL of this mixture to each of the ligation mixtures
*Two PCRs per colony:
*heat shocked for 2 min
**sbb18:
*added 100ul 2YT
***ca998+G00101
*rescued for 1.5 hours
***TH313+igemTen10
*plated 100ul of iGEM10_146(lifact ffGFP) assembly on CA
**iGEM10_004:
*plated 100ul of iGEM10_149(lifact RFP) assembly on CA
***ca998+G00101
***TH313+igemTen10
**iGEM10_005:
***ca998+G00101
***MLL2F+igemTen10
*TH313 is a fwd primer out of AmpR and MLL2F is a fwd primer out of CamR
*I swiped each colony on an ACK plate to check for cotransformation
<br>
''Gel Results''<br>
[[image:ConorGel55.jpg|400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lanes'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Checking'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable Lanes'''
|-
| 1 to 4||sbb18||pMLL6-KA||part||ca998||G00101||599||1,2,3,4
|-
| 5 to 8||sbb18||pMLL6-KA||vector||TH313||igemTen10||1939||1,2,3,4
|-
| 9 to 16||iGEM10_004||pMLL9-CA||part||ca998||G00101||3771||none
|-
| 17 to 24||iGEM10_004||pMLL9-CA||vector||TH313||igemTen10||1939||19,20,21,24
|-
| 25 to 32||iGEM10_005||pMLL8-KC||part||ca998||G00101||2354||25,29,30,32
|-
| 33 to 40||iGEM10_005||pMLL8-KC||vector||MLL2F||igemTen10||1808||33,35,37,38,40
|}


===Miniprep/Mapping iGEM10_006===
===Picking colonies of iGEM10_145===
*The colonies I picked yesterday were all on the wrong plasmid (AK not KA) and the internal plasmids did not seem to bind, but I will miniprep and digest, just in case the correct part is there within the wrong plasmid. The internal plasmids I used were long (designed for Gibson) and can be finicky.
*picked 4 green fluorescent colonies
<br><br>
*ran a colony PCR with ca998 and G00101
'''Digestion'''<br>
**expected size: 964
*standard Eco/Bam digestion (4uL H20, 1uL NEB2, 4uL DNA, 1uL each enzyme)
[[image:ConorGel99.jpg|200px]]
*digested all 5 minipreps
*no visible bands
''Gel Results''<br?
[[image:ConorGel56.jpg|400px]]
<br>
Expected Sizes:5734, 2868
*ladder warping makes it difficult to be precise, but if any lanes are correct, it is lane 3


==[[User:Conor McClune|Conor McClune]] 14:33, 14 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 17:30, 11 August 2010 (EDT)==
===Assembly of iGEM10_016===
===PCR magnification of Pcon part Bca1152===
'''Picking Colonies and Colony PCR'''
*template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
*picking 8 colonies
*conducted PCR with both Phusion and Expand master mixes
*swiping on cotransformation CKA plate
*recipe: 50uL MM (phusion or expand), 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
*dipping each colony in two col PCR mixes:
*both run on 2K55
**igemTen003+igemTen008
***checks the presence of both iGEM10_004 and iGEM10_005
***expected size:1461
**TH313+igemTen010
***checks order of Amp and Kan resistance genes in backbone
***desired (KA) size:  1939
***undesired (AK) size:  874
<br>
<br>
'''Gel Results:'''<br>
'''Gel Verification'''
[[Image:IMG 5339.JPG|400px]]<br>
*expected size:1079
*lanes 1-5: colonies 1-5 with igemTen003+igemTen008
*row 1 : 10uL phusion product
**checks the presence of both iGEM10_004 and iGEM10_005
*row 2 : 10uL expand product
**expected size:1461
**no bands visible in any lane
*lanes 6-10: colonies 1-5 with TH313+igemTen010
**checks order of Amp and Kan resistance genes in backbone
**desired (KA) size: 1939
**undesired (AK) size: 874
**all colonies have undesired antibiotic configuration (KA)
**no colonies were cotransformed


===Eco/Bam Transfer sbb18 into correct KA part===
[[image:ConorGel97.jpg|100px]]
'''Digestion:''' Standard Eco/Bam digestion of sbb18 from ''Romeo'', incubated for 30 minutes.<br><br>
*both products are the correct size, though yield is weak
*Phusion product looks best


'''Gel Purification:'''<br>
===Assembly of Lifact Payload Parts iGEM10_144-145===
sbb18:
====Digestion====
*desired fragment-408
*recipe: 4uL H20, 4uL DNA, 1uL NEB2, 1uL enzyme 1, 1uL enzyme 2
*undesired fragment-2868
*digested Bjh1883 and jtk2541 with Eco/Bgl
<br>
*digested both phusion and expand PCR products of Bca1152 with Eco/Bam
iGEM10_004:
*digested 4:53-
*desired fragment-3580
*undesired fragment-2868
<br>
iGEM10_005:
*desired fragment-2163
*undesired fragment-2868
<br>
I cut out the desired bands, dissolved them and zymo purified them.
<br><br>


'''Ligation:'''<br>
====Zymo Cleanup====
*master mix: 24uL H20, 4uL T4 buffer w/ATP, 4uL Ligase
*conducted a standard zymo cleanup of Bjh1883 and jtk2541
*8uL master mix added to 1uL of vector and 1uL part:
*conducted a small-fragment zymo cleanup of PCR products
**sbb18 with pMLL6
====Transformation====
**iGEM10_004 with pMLL9
*cell mixture: 200uL JTK030 cells, 30uL KCM
**iGEM10_005 with pMLL8
*added 70uL of this mixture to each of the ligation mixtures
<br>
*heat shocked for 2 min
*added 100ul 2YT
*rescued for 2.5 hours
*plated 100ul of iGEM10_145(ffGFP) assembly on Spec
*plated 100ul of iGEM10_144(RFP) assembly on CA
**since we were out of CA plates, I diluted 25uL 1000X Amp with 75uL H20 and plated this onto a Chlor plate


'''Transformation:'''<br>
==[[User:Conor McClune|Conor McClune]] 15:07, 10 August 2010 (EDT)==
*100ul Lefty pir+ mc1065
===Testing the SLD in our transposase parts iGEM10_007,13,20===
**added 30uL KCM to cells
Last night I picked colonies of each and grew them up in 6ml LB. iGEM10_007(piggybac) did not grow up because it was in the wrong color LB, but I repicked it at 11:30 and will test it later.
**add 70uL of cells/KCM to ligation mixture of sbb18
*100uL jtk030 cells
**added 50uL H20 and 40uL KCM
**added 70uL of cells to ligation mixtures for iGEM10_004 and iGEM10_005
<br>
*heat shocked cells for 90 seconds
*added 100uL 2YT to each of the 3 cultures
*rescued for 45 minutes
*plated:
**sbb18 on KA
**iGEM10_004 on CA
**iGEM10_005 on CK


==[[User:Conor McClune|Conor McClune]] 15:45, 13 July 2010 (EDT)==
iGEM10_013(sleeping beauty) and iGEM10_020(Tn5):
===Assembly of iGEM10_006===
*split each culture into two aliquots of 3mL - one with 3uL atc, one with nothing added
'''Digestion'''<br>
*placed these cultures on the incubator at 12:05
*iGEM10_005 #3: 4uL DNA, 4uL H2O, 1uL NEB2, 1uL Xhol, 1uL Bam
*iGEM10_004 #4: 4uL DNA, 4uL H2O, 1uL NEB2, 1uL Xhol, 1uL Bgl
*incubated from 11:19-12:25
<br>
'''Gel Purification'''
*iGEM10_004 #4:
**desired fragment:4841
**other fragment:1475
*iGEM10_005 #3:
**desired fragment:3629
**other fragment:1196
[[image:ConorGel54.jpg|400px]]
<br> I gel purified the desired fragments and zymo purified them. I accidentally used double the water (17uL), so my zymo is a little dilute and I had to modify the ligation procedure<br><br>
'''Ligation'''
*2uL digested iGEM10_004 #4
*2uL digested iGEM10_005 #3
*4uL H20
*1uL T4 ligase buffer with ATP
*1uL T4 Ligase
<br>
'''Transformation'''
*20uL KCM added to 100uL JTK030 comp cells
*all of ligation mixture added to these cells
*sat on ice for 10 minutes
*heat shocked at 42 for 90 seconds
*rescued at 37 on shaker form 2:56-4:02
*plated entire solution on KA plate


==[[User:Conor McClune|Conor McClune]] 14:14, 12 July 2010 (EDT)==
results at 1:05: no lysis
===Eco/Bam Transfering Basic parts to correct KA vector===
*used Tim's digested parts and Amy's new digest pMLL6-KA
*I eco/bam digested sbb42 for 1hr and Bjh2245 for 30 min
*created ligase MM:
**130uL H20
**20uL T4 ligase buffer
**20uL Eco/Bam digested pMLL6-KA
*added 8.5uL MM to 1uL of each of the following Eco/Bam digested parts:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Ebid'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Strain'''
|-
| 1||2||sbb09||R
|-
| 2||4||sbb07||R
|-
| 3||5||sbb06||R
|-
| 4||7||sbb04||R
|-
| 5||11||Bjh1906||R
|-
| 6||12||sbb19||R
|-
| 7||19||sbb12||R
|-
| 8||22||sbb10||R
|-
| 9||23||sbb42||R
|-
| 10||24||Bjh2294||R
|-
| 11||na||Bjh2245||R
|-
| 12||7||sbb04||L
|-
| 13||12||sbb19||L
|-
| ||||||
|-
| 15||22||sbb10||L
|-
| 16||23||sbb42||L
|}
*Transformed into the appropriate righty or lefty mc1061 pir+ strain


===Restriction Mapping===
results at 2:05: no lysis
*digestion master mixes- 4 H2O: 1 NEB2 : 0.5 Enzyme1 : 0.5 Enzyme1


==[[User:Conor McClune|Conor McClune]] 17:19, 9 August 2010 (EDT)==
===Self lysis in Varied Media:Results===
Procedure:
*Grew up 10mL of two colonies off the plates for igem10_020, 013, and 131
*Used 8mL of those cells by filling 4 collection tubes with 2mL of cells. Spun them down and disposed of supernatant.
*Resuspended one of each in TB, LB, Sea water, and Transposase Buffer.
*Divided each of the 12 2mL sample into two wells on a 24 well plate.
*Added 1uL of atc to one of the 1mL samples from each 2mL sample. See detailed layout of plate above.
*Moved plate to 37deg shaker. Let sit for 1hour.
*Transfered 200uL of cells from each well to an eppendorph tube and spun down the cells. Transfered supernatant to Zymo Columns.
*Performed a zymo. Note: used 800ul of ADB to meet the necessary 1:4 ratio. Eluted with 10uL.
*Put tubes on ice. Transformed a huge batch of Righty cells (pir +). Added 70uL of cells to each tube.
*Heat shocked, added 100uL of 2YT, let rescue for ~10min before realizing the plasmids are AC and I could just plate on Amp.
*Plates are currently stored in incubator labeled 1-24 according to the layout above.
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_20 (atc)'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_20 (no atc)'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_013 (atc)'''
| align="center" style="background:#f0f0f0;"|'''Expected Sizes'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_013 (no atc)'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_131 (atc)'''
|-
| align="center" style="background:#f0f0f0;"|'''iGEM10_131 (no atc)'''
| 1||sbb10 #1||pMLL6-KA||Eco/Bam||2868, 1035||yes
|-
| 2||sbb10 #2||pMLL6-KA||Eco/Bam||2868, 1035||yes
|-
| 3||sbb10 #3||pMLL6-KA||Eco/Bam||2868, 1035||yes
|-
| 4||sbb10 #4||pMLL6-KA||Eco/Bam||2868, 1035||yes
|-
| 5||iGEM10_004 #1||pMLL8-KC||Eco/Bam||2662, 2163||failed digest (righty)
|-
| 6||iGEM10_004 #2||pMLL8-KC||Eco/Bam||2662, 2163||failed digest (righty)
|-
| 7||iGEM10_004 #4||pMLL8-KC||Eco/Bam||2662, 2163||failed digest (righty)
|-
| 8||iGEM10_005 #6||pMLL8-KC||Eco/Bam||2662, 2163||failed digest (righty)
|-
| 9||sbb04 #1||pMLL6-KA||Eco/Bam||2868, 1797||yes
|-
| 10||sbb04 #2||pMLL6-KA||Eco/Bam||2868, 1797||yes
|-
| 11||Bjh2294 #1||pMLL6-KA||Eco/Bam||2868, 2516||probably
|-
| 12||Bjh2294 #2||pMLL6-KA||Eco/Bam||2868, 2516||probably
|-
|-
| 13||iGEM10_005 #2||pMLL9-CA||Eco/Xho||3227, 1598||yes
| Transposase Buffer||32||5||7||8||very high density||high density 
|-
|-
| 14||iGEM10_005 #3||pMLL9-CA||Eco/Xho||3227, 1598||yes
| ASW||70||135||35||49||high density||higher density
|-
|-
| 15||iGEM10_005 #4||pMLL9-CA||Eco/Xho||3227, 1598||yes
| LB||490||146||119||105||high density (almost lawn)||high density (almost lawn)
|-
|-
| 16||iGEM10_005 #8||pMLL9-CA||Eco/Xho||3227, 1598||yes
| TB||234||158||73||122||0||high density (almost lawn)
|}
|}
[[image: ConorGel52.JPG|400px]]
 
<br>
==[[User:Conor McClune|Conor McClune]] 14:31, 5 August 2010 (EDT)==
<br>iGEM10_004 failed to digest because Bam does not digest righty-methylated parts. I am redoing it with an Eco/Xho digest:<br>
===Transposase Assay Results===
[[image: ConorGel53.jpg|400px]]<br>
No growth on any plates
===His-tag addition to Transposases===
====PCR Isolation of Transposases====
The following three pcr's were run on 2K55:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Name'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Target Part'''
| align="center" style="background:#f0f0f0;"|'''Expected Sizes'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
|-
| 1||iGEM10_004 #1||pMLL8-KC||Eco/Xho||4850, 1466||too faint to see
|-
|-
| 2||iGEM10_004 #2||pMLL8-KC||Eco/Xho||4850, 1466||too faint to see
| 1||sbb10||KA||<SB100x!||ca998||igemTen048
|-
|-
| 3||iGEM10_004 #4||pMLL8-KC||Eco/Xho||4850, 1466||looks good
| 2||sbb04||KA||<piggyBac!||ca998||igemTen050
|-
|-
| 4||iGEM10_005 #6||pMLL8-KC||Eco/Xho||4850, 1466||looks good
| 3||iGEM10_055||KA||<tn5>{<NIS!}||ca998||igemTen051
|}
|}


===Eco/Bgl/Bam assembly of iGEM10_006, iGEM10_010, iGEM10_011===
==[[User:Conor McClune|Conor McClune]] 14:30, 4 August 2010 (EDT)==
'''Digestion'''
===Transposase Assay: Testing Sleeping Beauty and Tn5 in NEB2+MgCl2, varying [DNA] and activity time===
*digestion master mixes- 4 H2O: 1 NEB2 : 0.5 Enzyme1 : 0.5 Enzyme1
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Colony # or letter'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Desired Frag'''
| align="center" style="background:#f0f0f0;"|'''Other Fragment'''
|-
| iGEM10_005||pMLL8-KC||3||Eco/Bam||2163||2662
|-
| sbb10||pMLL6-KA||1||Eco/Bam||1035||2868
|-
| iGEM10_008||pMLL5-CK||d||Eco/Bam||1066||2662
|-
| iGEM10_004||pMLL9-CA||2||Eco/Bgl||6307||9
|-
| iGEM10_009||pMLL4-AC||b||Eco/Bgl||3015||9
|-
| Bjh2294||pMLL6-KA||1||Eco/Bgl||5375||9
|}
*I gel purified all the desired fragments
*note, there was no clearly visible band at 6307 in lane 4, but I cut out a larger area. The above restriction digests of iGEM10_004 suggest that it may have been better to use colony #3 or 4, not #2, as I did.


==[[User:Conor McClune|Conor McClune]] 22:25, 11 July 2010 (EDT)==
Transposition Buffer: Added 1.0165mg to each mL of NEB2  (MgCl2 tetrahydrate is 203.3g/mol and we're attempting to copy a transposition buffer that contains 150mM of MgCl2). We added 36.6mg of MgCl2 to 32.4mL of water and 3.6mL of 10xNEB2 to make '''150mM MgCl2 NEB2'''. We also added 5 drops of diluted HCl (tube labeled diluted HCl... we made it my dipping a pasteur pipette in the 12M HCL and then in 50mL of mgH20) to bring the pH from 7.8 to 7.6 or so.
===Col PCR results of picking iGEM10_004, iGEM10_005, sbb04 and Bjh2294===
[[image:ConorGel50.jpg|400px]]
*lanes 1-8 : iGEM10_004
**expected size:3771
**wrong size, most lanes seem to match the size of iGEM10_005 instead
*lanes 9-16 : iGEM10_005
**expected size:2354
** none are correct size, though lanes 1,2,4,5,6 match the size of iGEM10_004
*lanes 17-20 : sbb04
**expected size:1988
**all 4 lanes look good
*lanes 21-24 : Bjh2294
**expected size:2707
**all 4 lanes look good
Note: lanes 17-24 and final ladder are somewhat warped due to the plastic object I used to hold the gel
[[image:ConorGel51.jpg|200px]]


I must have switched the plates for iGEM10_005 and iGEM10_004 when I was picking, so I have the correct parts under the wrong labels. From this point one I will switch the iGEM10_005 and iGEM10_004 back.
Spun down all 5mLs of cells and 1uL of control cells (no ara). Resuspended in NEB2+MgCl2 buffer. Transferred control into a tube. Combined all 5mL resuspended cells into one tube, and then separated into 5 tubes.  


==[[User:Conor McClune|Conor McClune]] 20:15, 10 July 2010 (EDT)==
Added 1uL of atc and 10uL of arabinose to each mL of cells at 1:05pm. Put in 37deg shaker.
=== Replenish stcoks of sbb10===
*miniprepped all 4 colonies I grew up (though colony #1 looks best)


=== Replenish stocks of sbb04 and Bjh2294 (Self Lysis Device)===
Started adding DNA at 1:40pm. For DNA, we mixed two minipreps of 9145-1144. Well, we mixed them after adding DNA from one of the MPs to the 1uL tubes and the 13 .5uL tube (Whoops).
*picked 4 colonies of each and transferred to KA LB


===Assembly of iGEM10_004, iGEM10_005 and iGEM10_011===
Somehow, we ran out of MP, so we're only adding up to 10uL of DNA.
*no colonies grew for iGEM10_011, but a few grew up on the plates for iGEM10_004 and iGEM10_005


Time points: <br>
.5hr- 2:10pm <br>
1hr- 2:40pm <br>
1.5hr- 3:10pm <br>
2hr- 3:40pm


Transform into TG1 cells. We mixed 11 tubes of TG1 cells together in a Falcon tube.


==[[User:Conor McClune|Conor McClune]] 15:02, 9 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 16:53, 3 August 2010 (EDT)==
===Retransform Bjh2294 (self lysis device) and sbb04 for stock replentishment===
===Assembly of iGEM10_007===
*used Assembly teams methylated, 1:2 diluted stocks
The assembly of this part is being put on pause until it is confirmed that the identical part iGEM10_131 is in fact incorrect. The plate with colonies is in the refrigerator.
*transformed 4uL of of each miniprepped DNA into 100uL jtk030 comp cells (w/ 20uL KCM).
*rescued for 1hr at 37*C
*plated entire solutions onto KA plates


=== Replenish stocks of sbb10===
===Analytical PCR of iGEM10_131===
*picked 4 colonies from yesterdays transformation and placed in 3uL KA LB
Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
*set up a Col PCR with ca998 and G00101
*run on 4K45
'''Col PCR results:'''<br>
*expected size:1226
[[image:ConorGel49.jpg|400px]]<br>
''Results:''<br>
Colony one looks great, other colonies show faint lines.
 
=== Assembly of iGEM10_004, iGEM10_005, iGEM10_011===
'''Digestion'''
*made lefty and righty digestion MM
*digested the following parts:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''MM'''
| align="center" style="background:#f0f0f0;"|'''Desired Fragment'''
| align="center" style="background:#f0f0f0;"|'''Other Fragment'''
|-
| 1||sbb04||pMLL6-KA||L||3395||1270
|-
| 2||iGEM10_003b||pMLL5-CK||L||2539||1064
|-
| 3||iGEM10_008d||pMLL5-CK||L||2532||1196
|-
| 4||iGEM10_002d||pMLL4-AC||R||1430||1681
|-
| 5||iGEM10_009b||pMLL4-AC||R||1343||1681
|}
*I gel purified the desired bands
*Amy already had gel purified a lefty (xho/bam) digest of Bjh2294
'''Ligation'''
*I did standard ligations to make the following parts:
**sbb04 + iGEM10_002 = iGEM10_004
**iGEM10_003 + Bjh2294 = iGEM10_005
**iGEM10_008 + Bjh2294  = iGEM10_011
'''Transformation'''
*I transformed iGEM10_004 and iGEM10_011 into righty pir+ mc1061
**these I plated on CA plates after an hour's rescue
*I transformed iGEM10_005 into lefty pir+ mc1061
**these I plated on a KC plate after an hour's rescue
==[[User:Conor McClune|Conor McClune]] 14:03, 8 July 2010 (EDT)==
===Assembly of iGEM10_007 and iGEM10_013===
====Restriction Digest of iGEM10_004,iGEM10_005 and iGEM10_011====
Samples checked: iGEM10_004#7 ,iGEM10_005#1 and iGEM10_011#1 <br>
Well contents:
*12uL miniprepped DNA
*2uL BamHI
*2uL EcoR1
*1.5 NEB2
incubated at 37 for 1hr
'''Gel Results:'''<br>
[[image:ConorGel47.jpg|400px]]<br>
GEM10_004 #7:
*expected sizes:2756,3580
*smeared, but does not look like there are two bands
iGEM10_005 #1:
*expected sizes:2662,2163
*smeared, but it looks like there is an incorrect band around 1kb
iGEM10_011 #1:
*expected sizes:3573,2736
*smeared, second band possibly present, though faint
===Analytical PCR of iGEM10_004,iGEM10_005 and iGEM10_011===
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Part Checked for'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable Band?'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_004 #7||iGEM10_003||ca998||igemTen008||1164||yes, but also a band at 300bp
|-
| 2||iGEM10_004 #7||Bjh2294||igemTen007||G00101||2607||no
|-
| 3||iGEM10_005 #1||sbb04||ca998||igemTen004||1888||very faint band
|-
|-
| 4||iGEM10_005 #1||iGEM10_002||igemTen003||igemTen006||366||no
| 1||iGEM10_103||ca56||igemTen004||2045||yes
|-
|-
| 5||iGEM10_011 #1||iGME10_008||ca998||igemTen020||1157||yes, but also a band at 400bp
| 2||iGEM10_103||ca56||igemTen006||2411||yes
|-
|-
| 6||iGEM10_011 #1||Bjh2294||igemTen019||G00101||2607||no
| 3||iGEM10_103||ca56||igemTen008||3475||no
|}
|}
*Well contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
[[image:ConorGel96.jpg|400px]]
*run on program 3K45
[[image:ConorGel48.jpg|400px]]<br>
''Results'':
*bands in lanes 1,3,5, but not 2,4,6, indicating that none of the parts were assembled correctly. It looks like there is only the lefty half of each part in these plasmids.
====Remake of basic part Sbb10 from 140L stocks====
I have not been able to digest it and the Robot Assembly team has never been able to successfully use it in an assembly, so I am going to re-eco/bam it from a BioE 140L stock.


''Digest:'' Well contents: 4uL H20, 4uL stock sbb10, 1.1uL NEB2, 1uL Eco, 1uL Bam. Digested for 1hr.
===Preparation of Bacterial Stock Choano Food===
<br><br>
*Grew up 400mL MC1061 overnight on 37*C shaker
''Ligation:'' Ligated into pMLL6 using standard procedure.<br><br>
*split into two centrifuge bottles and centrifuged at 4200rpm for 14 minutes
''Transform:''Transformed ligation mixture into 100uL jtk030 comp cells (w/ 20uL KCM).
*poured off supernatant
*Rescued for 1.5 hour.
*washed each pellet with 10mL artificial sea water (ASW)
*plated entire culture on KA plate
*poured off ASW
*added 10mL ASW to each bottle and resuspended pellets
*centrifuged at 4200rpm for 14 minutes
*poured off supernatant
*resuspended pellets
*combined the contents of the two bottles
*created 2uL aliquots
*


==[[User:Conor McClune|Conor McClune]] 15:08, 7 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 14:39, 2 August 2010 (EDT)==
===2AB assembly of iGEM10_007 and iGEM10_013===
====Failure of iGEM10_010====
Due to all the cotransformations I am getting, I am going to gel purify the digestion fragments
*sbb10:
**digested with Xhol/BamH1
**expected sizes: 2633*, 1026
*iGEM10_009:
**digest with Xhol/BglII
**expected sizes: 1343*, 1681
Gel Results of yesterdays digestions: <br>
[[image:ConorGel44.jpg|400px]]<br>
*I gel purified the desired fragment of iGEM10_009, but it seems sbb10 was not digested well, so I will redo the digestion


====Digestion====
===Miniprep of iGEM10_103===
I will be redoing the digestion of sbb10 while also digesting iGEM10_004, iGEM10_005, iGEM10_011 for restriction mapping.<br><br>
The bacterial growth was smaller than usual, so I eluted with only 25uL H20, in order to avoid having a small concentration of DNA. I miniprepped all 6 picked clones of iGEM10_103.
'''Sbb10'''
*3uL H20
*8uL (1/2 diluted) miniprepped DNA (lefty methylated)
*1.5uL NEB2 + ATP
*1uL Xhol
*1uL BamH1
[[image:ConorGel46.jpg|400px]]<br>
*the part is still not digested. potential problems:
**bad enzyme?
<br>
'''Restriction Digests'''
*created 24uL Eco/Bam MM
*added 6uL MM to 4uL of iGEM10_004 #7, iGEM10_005 #1, iGEM10_011 #1
Results:<br>
[[image:ConorGel45.jpg|400px]]<br>
iGEM10_004 #7: 2756,3580
*expected sizes:
iGEM10_005 #1:
*expected sizes:2662,2163
iGEM10_011 #1:
*expected sizes:3573,2736
'''Results'''
*unclear, though only one band seems visible. I will rerun the digestion with a higher volume of DNA


==[[User:Conor McClune|Conor McClune]] 17:11, 6 July 2010 (EDT)==
====Restriction Mapping====
===Manual Assembly of iGEM10_004 ,iGEM10_005 ,iGEM10_010 and iGEM10_011===
*Digested 4uL of each miniprep with Xho/Bgl. Incubated from 11:30-12:15. Froze from 12:15-1:15.
'''Picking Colonies'''
*expected fragments: 6995 (desired fragment), 1607 (vector fragment)
*picked 8 colonies of each transformation
[[image:ConorGel93.jpg|400px]]
*set up Col PCR and swiped cotransformation plate
*no bands visible
Col PCR results:<bR>
*I want to gel purify the larger of the two fragments, so I cut out the entire 7kb section of all six rows, hoping to capture even a low concentration of the correct piece.
*note, all the PCR results seem to be wrong, but many are off by the same amount, so perhaps the ladder is off<br>
lanes 1-8:iGEM10_004 - expected size 3771
<br>lanes 10-17: iGEM10_005 - expected size 2354 <br>
[[image:ConorGel41.jpg|400px]]
*correct bands:
**iGEM10_004: lanes 4-7, with 7 being the strongest
***6 is cotransformed
***miniprep: 4,5,7
**iGEM10_005: lanes 1-3, 5-8
***none are cotransformed
***miniprep 1,2,3
<br><br>
lanes 1-8:iGEM10_010 - expected size 1505 <br>
[[image:ConorGel42.jpg|400px]]
*correct bands:
**none - 100% cotransformed
<br><br>
lanes 1-8:iGEM10_011 - expected size 3764 <br>
[[image:ConorGel43.jpg|400px]]
*correct bands:
**lanes 1,3 (neither cotransformed)


===Transformation of Bjh2294 to replenish stock===
*yesterday's transformation failed, because there were no colonies
*I will redo the transformation today
Procedure:
*add 50uL H20, 30uL KCN and 2uL DNA to 150uL mc1061 competent cells
*keep on ice for 20 min
*heat shock for 90 sec at 42*C
*mixed with 200uL 2YT and rescue for 1hr
*plate on KA plate


==[[User:Conor McClune|Conor McClune]] 18:14, 5 July 2010 (EDT)==
====Analytical PCR====
===2AB Assembly of Parts iGEM10_007 and iGEM10_013===
Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
===Digestion for Restriction Mapping and Assembly===
*run on 4K45
*made 7uL lefty and righty digestion master mix
*added made double the digestion solution for parts we constructed, so I would be able to run a restriction mapping gel with half:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''MM'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Volume MM'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable Clones'''
|-
|-
| 1||sbb04||7-H1||L||7
| 1||iGEM10_103||ca998||igemTen004||1859||1-3,5,6
|-
|-
| 2||iGEM10_003b||||L||14
| 2||iGEM10_103||ca998||igemTen006||2225||1-3,5,6
|-
|-
| 3||sbb10||22-A3||L||7
| 3||iGEM10_103||ca998||igemTen008||3289||none
|-
| 4||iGEM10_008d||||L||14
|-
| 5||iGEM10_002d||||R||14
|-
| 6||Bjh2294||24-G10||R||7
|-
| 7||iGEM10_009b||||R||14
|-
| 8||Bjh2294||24-G10||R||7
|}
|}
*incubated wells at 37 for 1hr
*zymo purified each of the wells
*used one uL of each purified digest for ligation
**combined wells:
***1 with 5 (to make iGEM10_004)
***2 with 6(to make iGEM10_005)
***3 with 7(to make iGEM10_010)
***4 with 8(to make iGEM10_011)
<br>
'''Restriction Digest'''
*digested with lefty MM (Xhol/BamHI)
**iGEM10_003b - expected sizes:2671,1064
**iGEM10_008d - expected sizes:2664,1064
*digested with righty MM (Xhol/BglII)
**iGEM10_002d - expected sizes:1681, 1430
**iGEM10_009b - expected sizes:1681, 1343
[[image:ConorGel40.jpg|400px]]<br>
''Results'': The strongest bands fit the desired bands. It looks like all these parts are good, though there are some other junk bands.


===Retransformation of Bjh2296===
'''Gel Pictures'''<Br>
*our first stock of Bjh2296 was getting low, so I retransformed it in mc1061
*ca998 + igemTen004
*note: 2YT used for rescue may be contaminated
[[image:ConorGel94.jpg|200px]]
*plated on KA plate
*ca998 + igemTen006 (lanes 1-6) and ca998 + igemTen008 (lanes 7-12)
[[image:ConorGel95.jpg|200px]]


==[[User:Conor McClune|Conor McClune]] 14:45, 4 July 2010 (EDT)==
'''Results'''<br> Either a part is missing around the 3'TerminalRepeat, or the pcr is bad. I will repeat it tomorrow
===2AB construction of iGEM10_007 and iGEM10_013===
'''Results from yesterdays Col PCR of iGEM10_004 and iGEM10_011'''
*iGEM10_004 - expected size: 3771
[[image:ConorGel38.jpg|400px]]


*iGEM10_011 - expected size: 3764
===Assembly of iGEM10_007===
[[image:ConorGel39.jpg|400px]]
====Digestion====
*I used a stored, previously digested version of iGEM10_001
*I used the above Xho/Bgl digestion of iGEM10_103 (aka iGEM10_006)


'''Results:'''<br>
====Ligation====
None of the col PCR fragments are the correct size for either of the parts.
*Recipe: 7uL iGEM10_103 purified digest, 1uL iGEM10_001 purified digest, 1uL T4 Buffer, 1uL T4 ligase
<br>
*incubated on desk from 2:55-3:25
Next Steps:
*do restriction digest to make sure the parts I am combining are correct
*redo 2AB assembly


==[[User:Conor McClune|Conor McClune]] 16:41, 3 July 2010 (EDT)==
===Transformation===
===2AB construction of iGEM10_007 and iGEM10_013===
*cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
'''Picking colonies'''<br>
*added 70uL of this cell mixture to the ligation mixture
*picked 8 colonies each for iGEM10_004 and iGEM10_011
*heat shocked for 90sec
*swiped each colongy on a AMp/Cam/Kan plate to check for cotransformation
*added 100uL 2YT and rescued for 1 hr
*ran 4K col pcr


==[[User:Conor McClune|Conor McClune]] 20:13, 2 July 2010 (EDT)==
===Transformed iGEM10_013 #5===
===2AB construction of iGEM10_007 and iGEM10_013===
*transformed into JTK030 cells for use in a transposase assay
*problem: yesterday I plated the two lefty cultures (containing parts iGEM10_004 and iGEM10_011) on KA plates rather than CA plates. Luckily I had saved the digestions from yesterday, so I religated  and retransformed
*cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
<br>
*added 70uL of this cell mixture to the ligation mixture
'''Picking Colonies'''
*heat shocked for 90sec
*picked 4 colonies each from the plates containing iGEM10_005 and iGEM10_010
*added 100uL 2YT and rescued for 1 hr
*swiped each colony on a cotransformation plate (Amp) after dipping in col PCR solution and 2uL KC media
''Col PCR''
*lanes 1-4: iGEM10_010 -expected size is 1500
*lanes 5-8: iGEM10_005 -expected size is 2350
[[image:ConorGel36.jpg|400px]]
*none of the colonies for either part are the right size. Both colonies need to be repicked<br>
'''Repicking Colonies'''
*picked 8 more colonies each of iGEM10_010 and iGEM10_005
*ran a col PCR on COL4K
*swiped each colony on an Amp plate to check for cotransformation
'''Results'''
*all 8 colonies of iGEM10_010 are cotransformed
*Gel results of col PCR for iGEM10_005 (expected size 2350):
[[image:ConorGel37.jpg|400px]]


===Choano Assays===  
===Grew up MC1061 cells to feed choanos in ASW===
*on Tuesday(3 days ago), I split choanos 1:15 into choano media under the following conditions:
4:00pm:  
**25*C(control)
*added 100uL MC1061 competent cells to 400mL LB
**37*C incubator
*placed on shaker at 37*C overnight
**37*C with shaking
**1mM NH4
**10mM NH4
*results were unconclusive, because even in the control choano density was too low for counts to be reliable, and florescence was too dim to see


==[[User:Conor McClune|Conor McClune]] 13:34, 1 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 23:44, 1 August 2010 (EDT)==
===2AB assembly for iGEM10_007 and iGEM10_013===
===Picking colonies for iGEM10_103 (iGEM10_006)===
{| {{table}}
*Picked 6 colonies - grew up in KA LB
| align="center" style="background:#f0f0f0;"|''''''
*Colony PCR
| align="center" style="background:#f0f0f0;"|'''Lefty'''
**primers: igemTen004 and ca56
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Righty'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Product'''
|-
| ||'''Part'''||'''Version'''||'''Part'''||'''Version'''||
|-
| ''iGEM10_007''||||||||||
|-
| ||sbb04||n/a||iGEM10_002||d||iGEM10_004
|-
| ||iGEM10_003||a||Bjh2294||n/a||iGEM10_005
|-
| ''iGEM10_013''||||||||||
|-
| ||sbb10||n/a||iGEM10_009||b||iGEM10_010
|-
| ||iGEM10_008||d||Bjh2294||n/a||iGEM10_011
|}
'''Digestion'''
*made up 35uL of lefty and righty MM
*mixed 4 uL of sbb04,iGEM10_003b,sbb10,iGEM10_008d each with 7uL lefty MM
*mixed 4 uL of iGEM10_002d,Bjh2294,iGEM10_009,Bjh2294 each with 7uL righty MM
*incubated for 1hr at 37*C
'''Ligation'''
*in each ligation: 6.5uL H2O, 1uL ligation buffer, 0.5uL ligase, 1uL lefty DNA, 1uL righty DNA
*Transformed into 70uL lefty and righty cells (parts 004 and 011 into lefty strain, and parts 005 and 010 into righty strain).
*plated lefty strains on CA and righty strains on KC plates
===Analytical PCR of iGEM10_020===
*Well contents: 33uL Taq/DMSO MM, 1uL template DNA, 1uL each oligo
*program: 4K55
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''reverse oligo'''
| align="center" style="background:#f0f0f0;"|'''best annealing temp'''
| align="center" style="background:#f0f0f0;"|'''parts tested for:'''
| align="center" style="background:#f0f0f0;"|'''expected size'''
|-
| 1||iGEM10_020a||ca56||igemTen022||55||igem10_001 and igem10_017||1800
|-
| 2||iGEM10_020a||igemTen021||G00101||55||igem10_016 and Bjh2294||3500
|-
| 3||iGEM10_020b||ca56||igemTen022||55||igem10_001 and igem10_017||1800
|-
| 4||iGEM10_020b||igemTen021||G00101||55||igem10_016 and Bjh2294||3500
|}
''Gel Results''<br>
[[image:ConorGel35.jpg|400px]]
*iGEM10_020b is complete

Latest revision as of 00:19, 17 October 2010

June Entries
July Entries


To Do:

Conor McClune 22:47, 16 October 2010 (EDT)

Transposase Assembly

Colony PCR analysis

Colony PCR failed: no bands

  • 171+174 (A-D):
    • lanes 1-4
    • expected size: 4615
    • good clones:
  • 171+175 (A-B):
    • lanes 5-6
    • expected size: 4416
    • good clones:
  • 170+175 (A-C):
    • lanes 7-9
    • expected size: 5265
    • good clones:
  • 170+174 (A-B):
    • lanes 10-11
    • expected size: 5464
    • good clones:
  • 170+173 (A-D):
    • lanes 12-15
    • expected size: 5471
    • good clones:

Conor McClune 18:11, 13 October 2010 (EDT)

Transposase assembly

Conducted the following digestions:

Lane Part Restr. Enzymes Parent Size Expected Fragments Acceptable? Gel Purified
1 163-5a Xho/Bam 6004 4730, 1270 yes
2 163-5b Xho/Bam 6004 4730, 1270 yes yes
3 163-10a Xho/Bam 5150 3880, 1270 no
4 163-10b Xho/Bam 5150 3880, 1270 no
5 163-17b Xho/Bam 5400 4100, 1270 yes yes
6 163-17a Xho/Bam 5400 4100, 1270 yes
7 ladder
8 163-17b Xho/Bgl 5400 1475, 3921 yes
9 2142-164a Xho/Bgl 5090 3408, 1680 yes yes
10 2142-164b Xho/Bgl 5090 3408, 1680 yes
11 2142-165a Xho/Bgl 5080 3401, 1680 yes yes
12 2142-165b Xho/Bgl 5080 3401, 1680 yes
13 2142-166a Xho/Bgl 4880 3200, 1680 yes
14 2142-166b Xho/Bgl 4880 3200, 1680 yes yes

Gel image:

preparation for plasmid payload cytometry

Self lysis test

  • started 2:00
  • checked at 4:30:
1 2 3 6 7
51 lysed lysed lysed barely lysed lysed
51 lysed lysed lysed lysed lysed


preparation of cells

  • scraped choanos and put 1ml in each test tube
  • added 40 uL bacteria to each test tube at 2:30
  • induced with arabinose fifteen minutes later (at 2:45)

Conor McClune 18:31, 12 October 2010 (EDT)

  • flow cytometry got no hits, because the choanos were in ASW, so the bacteria did not lyse

Conor McClune 18:31, 11 October 2010 (EDT)

preparation for plasmid payload cytometry

Self lysis test

  • started 2:15
  • checked at 4:15:
1 2 3 6 7
51 lysed lysed lysed lysed lysed
51 lysed lysed lysed lysed lysed


preparation of cells

  • scraped choanos and put 6ml in each test tube
  • added 240 uL bacteria to each test tube
  • induced with arabinose fifteen minutes later (at 2:30)

Conor McClune 19:02, 6 October 2010 (EDT)

Plasmid Delivery Assay

Plasmid Payload Numbers:

Number Part Antibiotic
1 pDYAL eGFP Amp
2 pDYAL eGFP Swar Amp
3 Clipit GFP Amp
4 M cherry Amp
5 Tera\'s α-tub GFP jtk159 Amp
6 Tera\'s efla GFP jtk160 Amp
7 PfuGWP GFP Amp
8 Tera\'s myo GFP jtk162 Amp
  • 51 and 52 refer to the Self-lysis/Vacuole-buster devices iGEM10_051-052

Procedure

Choanos

  • aspirated two plates (15ml each) of choanos in ASW, resuspended each in 5mL of choano media
    • note: the choano media we planned on using was contaminated, so I had to use an older one (one that seemed to be lacking something because choanos have not grown very well in it)
  • scraped choanos, mixed the two plates (to get 10mL), aliquoted 500uL into each of 12 mini slide wells
  • added 20uL of bacteria to the wells (10:15am):
1+51 2+51 3+51 6+51 7+51 lifeact iGEM10_171 +51(color control)
1+52 2+52 3+52 6+52 7+52 white bacteria
  • mistake:added 0.5uL ATC to each well at 10:30am, instead of arabinose (SLD is under Pbad not Ptet)
  • correction: added 5uL arabinose to each well at 11:00am

Self lysis test

    • made two 2mL aliquots of each of the plasmid bacteria cultures listed above
    • added 2uL ATC (mistake) and 20uL of arabinose to one of each of the aliquots of each culture

Results

Self Lysis Test

  • after 5 hours, all 10 plasmid+SLD/VB cultures with arabinose had lysed.

Conor McClune 22:30, 26 September 2010 (EDT)

Eco/Bam transfer of iGEM10_171 (A) into Bca1256

  • transformed product into JTK159 and plated on spec

Re-Assembly of iGEM10_170

  • gel purified my previous digestions of iGEM10_159 and Bjh1906
  • ligated 4uL of each with 1uL t4 buffer and 1uL t4 ligase
  • transformed into JTK159 and plated on CA

Conor McClune 16:34, 23 September 2010 (EDT)

LifeAct Assembly

Of the colonies if iGEM10_170 and iGEM10_171 picked yesterday, colonies 5,8,12 and 15 were cotransformed

Miniprepped: Colonies 1,2,3,7 (iGEM10_170) and colonies 10,11,18, 20 (iGEM10_171) because they were the reddest

  • renamed these clones iGEM10_170 A-D and iGEM10_171 A-D, respectively
    • iGEM10_171A sequenced well, with one mutation in the promoter
    • all of iGEM10_170 A-D are missing the terminator

Conor McClune 19:55, 22 September 2010 (EDT)

LifeAct Assembly

There is growth on the plates I made yesterday for iGEM10_170-171, though the Red/green colonies on iGEM10_171 are very small

I picked 8 colonies for iGEM10_170 and 12 from iGEM10_171 (only the last 4 may be green AND red).

Ran a colony PCR, but it did not show much because the addition of the terminator is not visible on the gel. The gel for the last colonies

  • lanes 1-8: iGEM10_170
    • all but colony 8 look good
  • lanes 9-13: iGEM10_171
    • all look good, except that col PCR failed for colony 12

Conor McClune 18:18, 21 September 2010 (EDT)

Restriction digest (Eco/Bam)of iGEM10_159-160

  • used iGEM10_159C
  • expected band sizes: 2660, 1580

  • the bands look good for both (faintess can be attributed to a bad zymo-forgot to spin out solvent for 90 sec before adding water)

Assembly of iGEM

Digested:

  • Xho/Bam: iGEM10_159C and iGEM10_160
  • Xho/Bgl: Bjh1906

Ligated each of the iGEM parts to left of Bjh1906 to make iGEM10_170-171, respectively
Transformed into JTK159 cells and plated on CA

Conor McClune 19:59, 18 September 2010 (EDT)

LifeAct Assembly

Redigestion of iGEM10_156A and iGEM10_159D

(this is necessary because the previous version of

  • Both were digested with Xho/Bam for 30 min
  • Zymo'd
  • Ligation (5:00-5:30):
    • iGEM10_156A + Bjh1881
    • iGEM10_159D + Bjh1906 (terminator)
    • iGEM10_159A + Bjh1906 (terminator)

Results: no growth for the iGEM10_159 + Bjh1906 assemblies, but one red/green colony for iGEM10_156A + Bjh1881 = iGEM10_160

  • colony PCR of iGEM10_160:

  • expected band : 1800bp, the band looks good

Remaking iGEM10_161 and iGEM10_162

(This is necessary because I had mislabelled iGEM10_157-158)
yesterday:

  • ligated the predigested combinations:
    • iGEM10_157 + Bjh1881
    • iGEM10_158 + Jtk2541

today:

  • transformed both ligations into JTK159 strain
  • plated on CK

Results: no growth, failed assembly

Conor McClune 18:38, 17 September 2010 (EDT)

LifeAct Assembly

Restriction digest of iGEM10_159-162 with Eco/Bam

  • expected sizes for all bands: 2660, 1580
  • iGEM10_159: lanes 1-4
  • iGEM10_160: lanes 5-8
  • iGEM10_161: lanes 9-12
  • iGEM10_162: lanes 13-16

Conor McClune 18:38, 14 September 2010 (EDT)

Assembly of LifeAct Payload

Potential Error discovered: plate with Eco/Bam iGEM157 and iGEM10158 were probably mislabelled since 157 was red and 158 was green (they should have been the green and red, respectively). This means that iGEM161-162 are probably just two copies of the same FP, one lifeact and one not.

Conor McClune 22:03, 12 September 2010 (EDT)

Lifeact Payload assembly

Digestion

  • Lefty digested: iGEM10_155-158
  • Righty digested: Bjh1881, Jtk2541
  • incubated 3:30-4:20

Ligation

  • ligating the following combinations:
    • iGEM10_155 + Jtk2541
    • iGEM10_156 + Bjh1881
    • iGEM10_157 + Bjh1881
    • iGEM10_158 + Jtk2541

Conor McClune 01:09, 10 September 2010 (EDT)

Lifact Assembly

  • Eco/Bam'd iGEM10_155-158 into pMLL9 (CA) (for continued assembly) and also into 1256 (Spec) (for assaying in choanos)

Conor McClune 01:28, 9 September 2010 (EDT)

Lifact Payload Assembly

  • I Eco/Bam digested iGEM10_155-158
  • Gel purified smaller fragment of each
  • meant to eco/bam into pMLL CA but we were out of the digested vector. I will have to digest this tomorrow

Conor McClune 18:04, 7 September 2010 (EDT)

Lifact Assembly

Colony PCR results

  • colony # corresponds to lane #
  • expected sizes:
    • lanes 1-4 Bca1237 + Bjh2252:1070
    • lanes 5-8 Bca1238 + Bjh2252:1070
    • lanes 9-4 Bca1237 + Bjh2251:1020
    • lanes 1-4 Bca1238 + Bjh2251:1020

  • note: broken well in lane 8, so contents were put in adjacent well

Results:

  • all lanes look good

Conor McClune 21:06, 5 September 2010 (EDT)

Lifact Payload Assembly

  • Ran a gel of Tahoura's PCR of Pcon parts Bca1237 and Bca1238:
    • image:
    • cut out 200-300bp range for both parts and small fragment gel purified (eluted with 8.5uL H20)
  • added 1uL each of NEB2, Bam and Eco to each of the purified products:
    • incubated at 37*C from 5:50-

Conor McClune 21:15, 13 August 2010 (EDT)

Assembly of iGEM10_146 and iGEM10_149

PCR magnification of Pcon part Bca1152

  • template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca9145
  • recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
  • run on 2K55


Gel Verification

  • expected size:206
  • gel purified the

The Expand seems to have worked best. It has a strong band at 200bp

Digestions

  • for Bca1152 (Pcon)-recipe:
    • 8uL of eluted PCR product
    • 1uL of NEB Buffer 2
    • 1uL EcoRI
    • 1uL BamHI
  • for Bjh2252(lifeact gfp) and Bjh2251 (lifeact rfp):
    • 14uL ddH2O
    • 2 uL NEB2+ATP Buffer
    • 1uL EcoRI
    • 1uL BamHI
    • 1uL miniprepped plasmid
  • incubated for 1hr at 37
  • zymo cleaned FP digests (eluted with 20uL), small fragment zymo cleaned Bca1152 (

Ligation

  • recipe:
    • 10uL vector digest (Bjh2251 or Bjh2251)
    • 7.5uL Bca1152
    • 2uL Ligase
    • 2uL T4 buffer +ATP
  • let sit on counter for 30 min

Transformation

  • cell mixture: 100uL Jtk030, 15uL KCM
  • 50uL of cell mixture added to each ligation mixture
  • let sit on ice for a few minutes
  • heat shocked at 42*C for 90 sec
  • added 100uL 2yt
  • rescued for 45 min
  • plated both on CA

Conor McClune 14:40, 12 August 2010 (EDT)

PCR magnification of Pcon part Bca1152

  • template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
  • recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
  • run on 2K55


Gel Verification

  • expected size:1079


The band looks too long

Assembly of iGEM10_146 and iGEM10_149

Digestion

  • standard Eco/Bgl digestion of Bjh2252 (GFP) and Bjh2251 (RFP)
  • 11:50-1:00
  • product was zymo purified

Ligation

  • recipe: 3uL H20, 1uL T4 buffer, 1uL T4 ligase, 1uL FP part (Bjh2252 or Bjh2251), 4uL yesterdays Eco/Bam digest of Expand PCR of Bca1152
  • incubated on desk from 11:50-12:30

Transformation

  • cell mixture: 200uL JTK030 cells, 30uL KCM
  • added 70uL of this mixture to each of the ligation mixtures
  • heat shocked for 2 min
  • added 100ul 2YT
  • rescued for 1.5 hours
  • plated 100ul of iGEM10_146(lifact ffGFP) assembly on CA
  • plated 100ul of iGEM10_149(lifact RFP) assembly on CA

Picking colonies of iGEM10_145

  • picked 4 green fluorescent colonies
  • ran a colony PCR with ca998 and G00101
    • expected size: 964

  • no visible bands

Conor McClune 17:30, 11 August 2010 (EDT)

PCR magnification of Pcon part Bca1152

  • template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
  • conducted PCR with both Phusion and Expand master mixes
  • recipe: 50uL MM (phusion or expand), 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
  • both run on 2K55


Gel Verification

  • expected size:1079
  • row 1 : 10uL phusion product
  • row 2 : 10uL expand product

  • both products are the correct size, though yield is weak
  • Phusion product looks best

Assembly of Lifact Payload Parts iGEM10_144-145

Digestion

  • recipe: 4uL H20, 4uL DNA, 1uL NEB2, 1uL enzyme 1, 1uL enzyme 2
  • digested Bjh1883 and jtk2541 with Eco/Bgl
  • digested both phusion and expand PCR products of Bca1152 with Eco/Bam
  • digested 4:53-

Zymo Cleanup

  • conducted a standard zymo cleanup of Bjh1883 and jtk2541
  • conducted a small-fragment zymo cleanup of PCR products

Transformation

  • cell mixture: 200uL JTK030 cells, 30uL KCM
  • added 70uL of this mixture to each of the ligation mixtures
  • heat shocked for 2 min
  • added 100ul 2YT
  • rescued for 2.5 hours
  • plated 100ul of iGEM10_145(ffGFP) assembly on Spec
  • plated 100ul of iGEM10_144(RFP) assembly on CA
    • since we were out of CA plates, I diluted 25uL 1000X Amp with 75uL H20 and plated this onto a Chlor plate

Conor McClune 15:07, 10 August 2010 (EDT)

Testing the SLD in our transposase parts iGEM10_007,13,20

Last night I picked colonies of each and grew them up in 6ml LB. iGEM10_007(piggybac) did not grow up because it was in the wrong color LB, but I repicked it at 11:30 and will test it later.

iGEM10_013(sleeping beauty) and iGEM10_020(Tn5):

  • split each culture into two aliquots of 3mL - one with 3uL atc, one with nothing added
  • placed these cultures on the incubator at 12:05

results at 1:05: no lysis

results at 2:05: no lysis

Conor McClune 17:19, 9 August 2010 (EDT)

Self lysis in Varied Media:Results

Procedure:

  • Grew up 10mL of two colonies off the plates for igem10_020, 013, and 131
  • Used 8mL of those cells by filling 4 collection tubes with 2mL of cells. Spun them down and disposed of supernatant.
  • Resuspended one of each in TB, LB, Sea water, and Transposase Buffer.
  • Divided each of the 12 2mL sample into two wells on a 24 well plate.
  • Added 1uL of atc to one of the 1mL samples from each 2mL sample. See detailed layout of plate above.
  • Moved plate to 37deg shaker. Let sit for 1hour.
  • Transfered 200uL of cells from each well to an eppendorph tube and spun down the cells. Transfered supernatant to Zymo Columns.
  • Performed a zymo. Note: used 800ul of ADB to meet the necessary 1:4 ratio. Eluted with 10uL.
  • Put tubes on ice. Transformed a huge batch of Righty cells (pir +). Added 70uL of cells to each tube.
  • Heat shocked, added 100uL of 2YT, let rescue for ~10min before realizing the plasmids are AC and I could just plate on Amp.
  • Plates are currently stored in incubator labeled 1-24 according to the layout above.
' iGEM10_20 (atc) iGEM10_20 (no atc) iGEM10_013 (atc) iGEM10_013 (no atc) iGEM10_131 (atc) iGEM10_131 (no atc)
Transposase Buffer 32 5 7 8 very high density high density
ASW 70 135 35 49 high density higher density
LB 490 146 119 105 high density (almost lawn) high density (almost lawn)
TB 234 158 73 122 0 high density (almost lawn)

Conor McClune 14:31, 5 August 2010 (EDT)

Transposase Assay Results

No growth on any plates

His-tag addition to Transposases

PCR Isolation of Transposases

The following three pcr's were run on 2K55:

Well Name Vector Target Part Fwd Oligo Rev Oligo
1 sbb10 KA <SB100x! ca998 igemTen048
2 sbb04 KA <piggyBac! ca998 igemTen050
3 iGEM10_055 KA <tn5>{<NIS!} ca998 igemTen051

Conor McClune 14:30, 4 August 2010 (EDT)

Transposase Assay: Testing Sleeping Beauty and Tn5 in NEB2+MgCl2, varying [DNA] and activity time

Transposition Buffer: Added 1.0165mg to each mL of NEB2 (MgCl2 tetrahydrate is 203.3g/mol and we're attempting to copy a transposition buffer that contains 150mM of MgCl2). We added 36.6mg of MgCl2 to 32.4mL of water and 3.6mL of 10xNEB2 to make 150mM MgCl2 NEB2. We also added 5 drops of diluted HCl (tube labeled diluted HCl... we made it my dipping a pasteur pipette in the 12M HCL and then in 50mL of mgH20) to bring the pH from 7.8 to 7.6 or so.

Spun down all 5mLs of cells and 1uL of control cells (no ara). Resuspended in NEB2+MgCl2 buffer. Transferred control into a tube. Combined all 5mL resuspended cells into one tube, and then separated into 5 tubes.

Added 1uL of atc and 10uL of arabinose to each mL of cells at 1:05pm. Put in 37deg shaker.

Started adding DNA at 1:40pm. For DNA, we mixed two minipreps of 9145-1144. Well, we mixed them after adding DNA from one of the MPs to the 1uL tubes and the 13 .5uL tube (Whoops).

Somehow, we ran out of MP, so we're only adding up to 10uL of DNA.

Time points:
.5hr- 2:10pm
1hr- 2:40pm
1.5hr- 3:10pm
2hr- 3:40pm

Transform into TG1 cells. We mixed 11 tubes of TG1 cells together in a Falcon tube.

Conor McClune 16:53, 3 August 2010 (EDT)

Assembly of iGEM10_007

The assembly of this part is being put on pause until it is confirmed that the identical part iGEM10_131 is in fact incorrect. The plate with colonies is in the refrigerator.

Analytical PCR of iGEM10_131

Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA

  • run on 4K45
Well Part Fwd Oligo Rev Oligo Expected Size Acceptable?
1 iGEM10_103 ca56 igemTen004 2045 yes
2 iGEM10_103 ca56 igemTen006 2411 yes
3 iGEM10_103 ca56 igemTen008 3475 no

Preparation of Bacterial Stock Choano Food

  • Grew up 400mL MC1061 overnight on 37*C shaker
  • split into two centrifuge bottles and centrifuged at 4200rpm for 14 minutes
  • poured off supernatant
  • washed each pellet with 10mL artificial sea water (ASW)
  • poured off ASW
  • added 10mL ASW to each bottle and resuspended pellets
  • centrifuged at 4200rpm for 14 minutes
  • poured off supernatant
  • resuspended pellets
  • combined the contents of the two bottles
  • created 2uL aliquots

Conor McClune 14:39, 2 August 2010 (EDT)

Miniprep of iGEM10_103

The bacterial growth was smaller than usual, so I eluted with only 25uL H20, in order to avoid having a small concentration of DNA. I miniprepped all 6 picked clones of iGEM10_103.

Restriction Mapping

  • Digested 4uL of each miniprep with Xho/Bgl. Incubated from 11:30-12:15. Froze from 12:15-1:15.
  • expected fragments: 6995 (desired fragment), 1607 (vector fragment)

  • no bands visible
  • I want to gel purify the larger of the two fragments, so I cut out the entire 7kb section of all six rows, hoping to capture even a low concentration of the correct piece.


Analytical PCR

Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA

  • run on 4K45
Well Part Fwd Oligo Rev Oligo Expected Size Acceptable Clones
1 iGEM10_103 ca998 igemTen004 1859 1-3,5,6
2 iGEM10_103 ca998 igemTen006 2225 1-3,5,6
3 iGEM10_103 ca998 igemTen008 3289 none

Gel Pictures

  • ca998 + igemTen004

  • ca998 + igemTen006 (lanes 1-6) and ca998 + igemTen008 (lanes 7-12)

Results
Either a part is missing around the 3'TerminalRepeat, or the pcr is bad. I will repeat it tomorrow

Assembly of iGEM10_007

Digestion

  • I used a stored, previously digested version of iGEM10_001
  • I used the above Xho/Bgl digestion of iGEM10_103 (aka iGEM10_006)

Ligation

  • Recipe: 7uL iGEM10_103 purified digest, 1uL iGEM10_001 purified digest, 1uL T4 Buffer, 1uL T4 ligase
  • incubated on desk from 2:55-3:25

Transformation

  • cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
  • added 70uL of this cell mixture to the ligation mixture
  • heat shocked for 90sec
  • added 100uL 2YT and rescued for 1 hr

Transformed iGEM10_013 #5

  • transformed into JTK030 cells for use in a transposase assay
  • cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
  • added 70uL of this cell mixture to the ligation mixture
  • heat shocked for 90sec
  • added 100uL 2YT and rescued for 1 hr

Grew up MC1061 cells to feed choanos in ASW

4:00pm:

  • added 100uL MC1061 competent cells to 400mL LB
  • placed on shaker at 37*C overnight

Conor McClune 23:44, 1 August 2010 (EDT)

Picking colonies for iGEM10_103 (iGEM10_006)

  • Picked 6 colonies - grew up in KA LB
  • Colony PCR
    • primers: igemTen004 and ca56
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