Berk2010-Conor: Difference between revisions

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[[Berk2010-Conor (June) | June Entries]]<br>
[[Berk2010-Conor (July) | July Entries]]
==To Do:==
==To Do:==
*split choanos Sun 7/20
*do Gibson PCR
**for assembly of iGEM10_007, iGEM10_013 and iGEM10_020
**use 1601(Jin's assembly vector) and the strain mc1061
**individually PCR parts for assembly
**gel purify PCR results
**parts remaining:
***iGEM10_001 w/ ca998 + 34
***B10sbb10 w/ 34+36
***iGEM10_009 w/ 35 + 18
***Bjh2294 w/  19+G00101
***Bjh2294 w/  23+G00101
==[[User:Conor McClune|Conor McClune]] 15:14, 21 June 2010 (EDT)==
===Gibson Assembly iGEM10_007, Attempt #2===
*cells grew up up on CA plate, no cells on negative control plates (K and Spec), besides the usual few tiny colonies on Spec
*picked 16 colonies:
**for 8 I did colony PCR using ca998 and G00101
**for 8 I did colony PCR with iGEM oligos 1 and 6
**growing up all 16 colonies in 1mL CA LB
====Colony PCR====
With primers ca998 and G00101:
*expected band size: 9000
*gel picture:
[[image:ConorGel18.jpg|400px]]
*Results:


With iGEM primers 1 and 6:
==[[User:Conor McClune|Conor McClune]] 22:47, 16 October 2010 (EDT)==
*expected band size: 2186
===Transposase Assembly===
*gel picture:
====Colony PCR analysis====
[[image:ConorGel17.jpg|400px]]
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:


===Manual Assembly of iGEM10_004 and iGEM10_005===
==[[User:Conor McClune|Conor McClune]] 18:11, 13 October 2010 (EDT)==
'''Digestion'''
*digested sbb04 and iGEM10_003 with lefty MM
*digested bjh2294 and iGEM10_002 with righty MM
*zymo cleaned all 4
*ligated sbb04 with iGEM10_002 (to make iGEM10_004) and bjh2294 with iGEM10_003 (to make iGEM10_005)
*transformed iGEM10_005 into lefty and plated on KC
*transformed iGEM10_004 into righty and plated on CA


==[[User:Conor McClune|Conor McClune]] 19:16, 20 June 2010 (EDT)==
===Transposase assembly===
===Gibson Chemistry===
Conducted the following digestions:
====Gibson Assembly of iGEM10_007 from June 18====
*Tim picked 10 colonies yesterday and grew them up overnight
*miniprepped and digested with EcoRI and Xho1 for restriction mapping
**expected fragment lengths: 9067 and 1196
[[Image:ConorGel16.jpg|400px]]
*none of these bands match the desired fragment lengths
*need to redo Gibson
====Gibson Assembly of iGEM10_007--Attempt #2====
*the 5uL DNA is made up of these volumes:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''PCR Purified'''
| align="center" style="background:#f0f0f0;"|'''Restr. Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Size'''
| align="center" style="background:#f0f0f0;"|'''Parent Size'''
| align="center" style="background:#f0f0f0;"|'''Band Brightness'''
| align="center" style="background:#f0f0f0;"|'''Expected Fragments'''
| align="center" style="background:#f0f0f0;"|'''Weight'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
| align="center" style="background:#f0f0f0;"|'''Volume added (uL)'''
| 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
|-
|-
| iGEM10_001||yes||1335|| bright||0.25||0.3125
| 3||163-10a||Xho/Bam||5150||3880, 1270||no||
|-
|-
| (B10sbb04)||yes||1788|| bright||0.25||0.3125
| 4||163-10b||Xho/Bam||5150||3880, 1270||no||
|-
|-
| iGEM10_002||yes||366||dim||0.5||0.625
| 5||163-17b||Xho/Bam||5400||4100, 1270||yes||yes
|-
|-
| iGEM10_003||yes||1064||dim||1||1.25
| 6||163-17a||Xho/Bam||5400||4100, 1270||yes||
|-
|-
| Self Lysis Device (Bjh2294)||yes||2507||medium bright||1||1.25
| 7||ladder||||||||||
|-
|-
| pBjh1601AC (vector)||yes||3183||medium bright||1||1.25
| 8||163-17b||Xho/Bgl||5400||1475, 3921||yes||
|-
|-
| ||||||||||
| 9||2142-164a||Xho/Bgl||5090||3408, 1680||yes||yes
|-
|-
| ||||||total||4||5
| 10||2142-164b||Xho/Bgl||5090||3408, 1680||yes||
|}
*added 15uL Gibson MM
*incubated at 50*C for 60 min
*Heat shock tranformation(see general protocol) with these volumes:
**200uL competent mc1061
**30uL KCN
**20uL gibson DNA
**after rescue, I plated 70uL each on AC, K and Spec plates (K and Spec are negative controls)
 
==[[User:Conor McClune|Conor McClune]] 13:54, 18 June 2010 (EDT)==
===Gibson Chemistry===
====Parts PCR====
'''RePCR of Bjh1601 AC on closest, black thermocycler'''
*Well contents: 33uL Phusion MM, 1uL each oligo (ss13r and igemTen009), .5uL template DNA
*run on program 4K55
[[image:ConorGel14.jpg|400px]]
*This confirms that the problem I was having was due to the program I was using in the iGEM folder on the white thermocycler.
*This band looks perfect (3100bp) and is even stronger than the original.
*I gel purified it
<br>
<br>
'''DpnI Digestion of Bjh1601 AC'''<br>
*started with 8.5uL zymo-purified Bjh1601 AC from above procedure
*added 0.5uL DpnI and 0.9uL NEB buffer 2
*incubated for 1hour at 37*C
<br>
<br>
'''PCR of Bjh2294 with fwd oligos 19 and 23"<br>
Well Contents: 50uL phusion MM, 0.5uL DNA template, 1uL each oligo
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Thermocycler Prgm'''
|-
|-
| 1||Bjh2294||Phusion||19||G00101||4K55
| 11||2142-165a||Xho/Bgl||5080||3401, 1680||yes||yes
|-
|-
| 2||Bjh2294||Phusion||23||G00102||4K55
| 12||2142-165b||Xho/Bgl||5080||3401, 1680||yes||
|-
|-
| 3||Bjh2294||Phusion||19||G00103||4K45
| 13||2142-166a||Xho/Bgl||4880||3200, 1680||yes||
|-
|-
| 4||Bjh2294||Phusion||23||G00104||4K45
| 14||2142-166b||Xho/Bgl||4880||3200, 1680||yes||yes
|}
|}
Gel image:<br>
[[image:ConorGel105.jpg|400px]]


''Gel Results'' (the lane numbers correspond the the well numbers above)
===preparation for plasmid payload cytometry===
<br>
====Self lysis test====
[[image:ConorGel15.jpg|400px]]
*started 2:00
*bands in lane 3 and 4 are strong and match the expected 2507bp
*checked at 4:30:
**I gel purified these bands
 
====Gibson Assembly of iGEM10_007====
Well contents:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| ||1||2||3||6||7
| align="center" style="background:#f0f0f0;"|'''Volume added (uL)'''
|-
| iGEM10_001||0.833
|-
|-
| <piggybac!(B10sbb04)||0.833
| 51||lysed||lysed||lysed||barely lysed||lysed
|-
|-
| iGEM10_002||0.833
| 51||lysed||lysed||lysed||lysed||lysed
|-
| iGEM10_003||0.833
|-
| Self Lysis Device (Bjh2294)||0.833
|-
| p1601AC (vector)||0.833
|-
| Gibson MM||20
|}
|}


*Incubated for about 30 minutes at 50*C before somebody pulled it off the thermocycler
*Incubated for an additional 30 minutes at 50*C
*Heat shock tranformation(see general protocol) with these volumes:
**200uL competent mc1061
**30uL KCN
**20uL gibson DNA
**plated 70uL each on AC, K and Spec plates (K and Spec are negative controls)


==[[User:Conor McClune|Conor McClune]] 13:54, 17 June 2010 (EDT)==
====preparation of cells====
===Gibson Chemistry===
*scraped choanos and put 1ml in each test tube
====Parts Preparation====
*added 40 uL bacteria to each test tube at 2:30
'''DpnI Digestion''' <br>
*induced with arabinose fifteen minutes later (at 2:45)
I added 0.85 uL NEB2 buffer and 0.5uL DpnI to my Bjh1601 AC PCR product (gel purified) in order to digest all remaining template DNA, which would interfere with the Gibson reacton. The mixture was placed in the incubator at 10:26 and will be ready at 11:26.
*11.26: Zymo Preparation
**during zymo purification, I accidentally added 200 uL N3j Qiagen Neutralization buffer, instead of PE buffer.
**attempt to recover DNA:
***added 200uL PE to N3/DNA solution
***spun through zymo column in 30sec
***added 200 ADB buffer to solution
***spun through same zymo column in 30 sec
***discarded liquid waste
***spun 200uL PE though zymo column in 15 sec
***discarded liquid waste
***spun 200uL PE though zymo column in 15 sec
***discarded liquid waste
***spun for 90 sec to remove any remaining PE buffer
***discarded waste
***added 17uL H20
***spun into a new eppendorf in 30 seconds
***run on gel <br>
[[image:ConorGel11.jpg|400px]] <br>
I cut out the band at 3kb and gel purified it. The band is not intense, but it does seem I was able to recover some of the DNA. I gel purified this DNA, but I would prefer to use the product of the below PCR, if it is successful.


'''RePCR of Bjh1601 AC'''
==[[User:Conor McClune|Conor McClune]] 18:31, 12 October 2010 (EDT)==
*Well contents: 33uL Phusion MM, 1uL each oligo (ss13r and igemTen009), .5uL template DNA
*flow cytometry got no hits, because the choanos were in ASW, so the bacteria did not lyse
*run on program 4K55
[[image:ConorGel13.jpg|400px]]
*These were the exact procedures I used to originally PCR pBjh1601AC, with one difference: the thermocycler I used (I used the white one on the back wall and ran the 4k55 program under the iGEM folder. Note to future self: do not use programs in this folder). I will repeat this on the same thermocycler


<br>
==[[User:Conor McClune|Conor McClune]] 18:31, 11 October 2010 (EDT)==
'''PCR of sbb10'''
===preparation for plasmid payload cytometry===
*Continued attempts to PCR sbb10
====Self lysis test====
*double checked oligos on ApE
*started 2:15
*created new 1:10 dilutions of oligos igemTen 14 and igemTen16
*checked at 4:15:
*in each well: 33uL polymerase MM, 1uL each of diluted igemTen 14 and igemTen16, 0.5 uL sbb10 from well A3 on Parts Plate 1
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| ||1||2||3||6||7
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
|-
| 1||sbb10||Phusion||igemTen14||igemTen16
|-
| 2||sbb11||Expand||igemTen14||igemTen16
|-
|-
| 3||sbb12||Phusion||igemTen14||igemTen16
| 51||lysed||lysed||lysed||lysed||lysed
|-
|-
| 4||sbb13||Expand||igemTen14||igemTen16
| 51||lysed||lysed||lysed||lysed||lysed
|}
|}
Wells 1,2 run on program COL2K<br>
Wells 3,4 run on program 2K45 <br>
[[image:ConorGel12.jpg|400px]]
*still no bands appear in the desired 260bp range.
*checked clotho --> the sequence we had entered for sbb10 on our "parts and data" spreadsheet was incorrect
*need to redesign primers igemTen14 and igemTen16
* I redesigned the primers


==[[User:Conor McClune|Conor McClune]] 13:52, 16 June 2010 (EDT)==
===Gibson Chemistry===
====Parts PCR====
''Gel Results from PCR of parts for assembly of iGEM10_013 and iGEM10_020''<br>
[[image:ConorGel9.jpg|400px]]


====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;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Number'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Antibiotic'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Size Expected'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
|-
| 1||B10sbb10||Expand||igemTen014||igemTen016||260||Very faint
| 1||pDYAL eGFP||Amp
|-
|-
| 2||iGEM10_009 A||Expand||igemTen015||igemTen018||310|| Yes
| 2||pDYAL eGFP Swar||Amp
|-
|-
| 3||iGEM10_008d ||Expand||igemTen017||igemTen020||1090|| Yes
| 3||Clipit GFP||Amp
|-
|-
| 4||iGEM10_017 A||Expand||ca998||igemTen022||1650|| Yes
| 4||M cherry||Amp
|-
|-
| 5||iGEM10_016a||Expand||igemTen021||igemTen024||890|| Yes
| 5||Tera\'s α-tub GFP jtk159||Amp
|-
|-
| 6||pB6 (BAC)||Expand||igemTen025||igemTen026||1000||Yes
| 6||Tera\'s efla GFP jtk160||Amp
|}
*Gel Purified lanes 2-5 (iGEM10_009 A,iGEM10_008d,iGEM10_017 A and iGEM10_016a)
*the results of lane six demonstrate that our genomic miniprep of pB6 was successful and has a high concentration of BAC DNA
 
====PCR of B10sbb10, Bjh2294 and pBjh1601AC====
*a matching rev backbone primer for pBjh1601AC was found: ss13r
*due to failure of previous pcr attempts for amplifying B10sbb10 and Bjh2294, I decided to try thermocycling at a lower temperature (45*C)
*two well strips:
**well contents: 33uL polymerase MM, 1uL each oligo, .5uL template DNA
**Strip 1: run on 4K45 (max temp=45*C)
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
|-
|-
| 1||B10sbb10||Phusion||igemTen014||igemTen016
| 7||PfuGWP GFP||Amp
|-
|-
| 2||B10sbb10||Expand||igemTen014||igemTen016
| 8||Tera\'s myo GFP jtk162||Amp
|-
| 3||Bjh2294||Phusion||igemTen007||G00101
|-
| 4||Bjh2294||Expand||igemTen007||G00101
|-
| 5||Bjh2294 (PCR Product)||Phusion||igemTen007||G00101
|}
|}
**Strip 2: run on 4K55(max temp=55*C)
 
*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):
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| 1+51||2+51||3+51||6+51||7+51||lifeact iGEM10_171 +51(color control)
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Size Expected'''
|-
| 1||B10sbb10||Phusion||igemTen014||igemTen016||260
|-
|-
| 2||pBjh1601||Phusion||igemTen009||ss13r||3183
| 1+52||2+52||3+52||6+52||7+52||white bacteria
|-
| 3||pBjh1601||Expand||igemTen009||ss13r||3183
|}
|}
*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
==[[User:Conor McClune|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
==[[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
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
[[image:ConorGel104.jpg|200px]]
*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
==[[User:Conor McClune|Conor McClune]] 18:18, 21 September 2010 (EDT)==
===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)


''Gel Results''
===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>
[[image:ConorGel10.jpg||400px]]
Transformed into JTK159 cells and plated on CA
{| {{table}}
 
| align="center" style="background:#f0f0f0;"|'''Lane'''
==[[User:Conor McClune|Conor McClune]] 19:59, 18 September 2010 (EDT)==
| align="center" style="background:#f0f0f0;"|'''Part'''
===LifeAct Assembly===
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
====Redigestion of iGEM10_156A and iGEM10_159D====
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
(this is necessary because the previous version of
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
*Both were digested with Xho/Bam for 30 min
| align="center" style="background:#f0f0f0;"|'''Size Expected'''
*Zymo'd
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
*Ligation (5:00-5:30):
|-
**iGEM10_156A + Bjh1881
| 1||B10sbb10||Phusion||igemTen014||igemTen016||260|| no
**iGEM10_159D + Bjh1906 (terminator)
|-
**iGEM10_159A + Bjh1906 (terminator)
| 2||B10sbb10||Expand||igemTen014||igemTen016||260|| no
 
|-
''Results:'' no growth for the iGEM10_159 + Bjh1906 assemblies, but one red/green colony for iGEM10_156A + Bjh1881 = iGEM10_160
| 3||Bjh2294||Phusion||igemTen007||G00101||2540|| Probably just template DNA
*colony PCR of iGEM10_160:
|-
[[image:ConorGel102.jpg|100px]]
| 4||Bjh2294||Expand||igemTen007||G00101||2540||no
*expected band : 1800bp, the band looks good
|-
 
| 5||Bjh2294 (PCR Product)||Phusion||igemTen007||G00101||2540|| yes
====Remaking iGEM10_161 and iGEM10_162====
|-
(This is necessary because I had mislabelled iGEM10_157-158)<br>
| 6||-||-||-||-||-||
yesterday:  
|-
*ligated the predigested combinations:
| 7||B10sbb10||Phusion||igemTen014||igemTen016||260|| no
**iGEM10_157 + Bjh1881
|-
**iGEM10_158 + Jtk2541
| 8||pBjh1601||Phusion||igemTen009||ss13r||3183|| yes
today:
|-
*transformed both ligations into JTK159 strain
| 9||pBjh1601||Expand||igemTen009||ss13r||3183||no
*plated on CK
|}
 
*none of the B10sbb10 PCR attempts worked
''Results:'' no growth, failed assembly
*I gel purified the desired bands in lanes 5 and 8:  Bjh2294 (PCR Product) and pBjh1601
 
==[[User:Conor McClune|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
[[Image:Conor 9-14-10.JPG|400px]]
 
[[Image:Conor 9-14-10 2.JPG|400px]]
 
==[[User:Conor McClune|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.''
 
==[[User:Conor McClune|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
 
==[[User:Conor McClune|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)
 
==[[User:Conor McClune|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


==[[User:Conor McClune|Conor McClune]] 14:00, 15 June 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 18:04, 7 September 2010 (EDT)==
===Gibson Chemistry===
===Lifact Assembly===
====Parts PCR====
====Colony PCR results====
''Gel Results from yesterday's Expand PCR''<br>
*colony # corresponds to lane #
*note: it appears some of well 6 (p1601ac) has evaporated. Perhaps the lid was not closed completely.
*expected sizes:
[[image: ConorGel6.jpg|400px]]
**lanes 1-4 Bca1237 + Bjh2252:1070
*gel looked messy, so I ran it again:
**lanes 5-8 Bca1238 + Bjh2252:1070
[[image: ConorGel7.jpg|400px]]
**lanes 9-4 Bca1237 + Bjh2251:1020
{| {{table}}
**lanes 1-4 Bca1238 + Bjh2251:1020
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_001 a||ca998||igemTen002||1360|| Yes
|-
| 3||iGEM10_001 b||ca998||igemTen002||1360||Yes
|-
| 5||iGEM10_001 c||ca998||igemTen002||1360||Yes
|-
| 7||<piggybac! (sbb04)||igemTen001||igemTen004||1820||Yes
|-
| 9||Self Lysis Device (Bjh2294)||igemTen007||G00101||2540||Very Faint
|-
| 11||1601AC plasmid||igemTen009||igemTen010||3220||No, not visible
|}
*Gel purified iGEM10_001-c (lane 5) and <piggybac!(sbb04) (lane 7)
*problem identified for PCR of p1601 AC : the reverse backbone oligo (igemTen10) does not match perfectly (it was originally designed for pMLL9-CA)


====RePCR of SLD and p1601 AC====
[[image:ConorGel101.jpg|400px]]
{| {{table}}
*note: broken well in lane 8, so contents were put in adjacent well
| align="center" style="background:#f0f0f0;"|'''Well'''
Results:
| align="center" style="background:#f0f0f0;"|'''Part'''
*all lanes look good
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
|-
| 1||Self Lysis Device(Bjh2294)||Phusion||igemTen007||G00101
|-
| 2||Self Lysis Device(Bjh2294)||Expand||igemTen007||G00101
|-
| 3||Self Lysis Device(Bjh2294)||Taq||igemTen007||G00101
|-
| 4||1601AC plasmid||Phusion||igemTen009||G00101
|-
| 5||1601AC plasmid||Expand||igemTen009||G00101
|-
| 6||1601AC plasmid||Taq||igemTen009||G00101
|}
Well contents: 33uL polymerase MM, 1uL each oligo, 0.5uL template DNA
*Thermocycler program 4K55
[[image: ConorGel8.jpg|400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
|| 1||Self Lysis Device(Bjh2294)||Phusion||igemTen007||G00101||2540||Very Faint, but visible
|-
| 2||Self Lysis Device(Bjh2294)||Expand||igemTen007||G00101||2540||No, nothing visible
|-
| 3||Self Lysis Device(Bjh2294)||Taq||igemTen007||G00101||2540||Very Faint, but visible
|-
| 4||1601AC plasmid||Phusion||igemTen009||G00101||3220|| No, nothing visible
|-
| 5||1601AC plasmid||Expand||igemTen009||G00101||3220||No, nothing visible
|-
| 6||1601AC plasmid||Taq||igemTen009||G00101||3220||No, nothing visible
|}
*I gel purified the bands at 2540 in lanes 1 and 3 (both are Bjh2294, Self Lysis Device)


====PCR of parts for assembly of iGEM10_013 and iGEM10_020====
==[[User:Conor McClune|Conor McClune]] 21:06, 5 September 2010 (EDT)==
Well contents: 33uL polymerase MM, 1uL each oligo, 0.5uL template DNA
===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-


{| {{table}}
==[[User:Conor McClune|Conor McClune]] 21:15, 13 August 2010 (EDT)==
| align="center" style="background:#f0f0f0;"|'''Well'''
===Assembly of iGEM10_146 and iGEM10_149===
| align="center" style="background:#f0f0f0;"|'''Part'''
====PCR magnification of Pcon part Bca1152====
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
*template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca9145
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
*recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
*run on 2K55
|-
<br>
| 1||B10sbb10||Expand||igemTen014||igemTen016
'''Gel Verification'''
|-
*expected size:206
| 2||iGEM10_009 A||Expand||igemTen015||igemTen018
*gel purified the
|-
The Expand seems to have worked best. It has a strong band at 200bp
| 3||iGEM10_008d ||Expand||igemTen017||igemTen020
|-
| 4||iGEM10_017 A||Expand||ca998||igemTen022
|-
| 5||iGEM10_016a||Expand||igemTen021||igemTen024
|-
| 6||pB6 (BAC)||Expand||igemTen025||igemTen026
|}


==[[User:Conor McClune|Conor McClune]] 15:13, 14 June 2010 (EDT)==
====Digestions====
===Gibson Assembly for iGEM10_007===
*for Bca1152 (Pcon)-recipe:
====Components====
**8uL of eluted PCR product
*Parts (assembled in this order):
**1uL of NEB Buffer 2
**iGEM10_001
**1uL EcoRI
**<piggybac!> (part name B10sbb04)
**1uL BamHI
**iGEM10_002
*for Bjh2252(lifeact gfp) and Bjh2251 (lifeact rfp):
**iGEM10_003
** 14uL ddH2O
**Self Lysis Device (part name Bjh2294)
** 2 uL NEB2+ATP Buffer
*Vector:
** 1uL EcoRI
**pBjh1601AC
** 1uL BamHI
====Parts PCR====
** 1uL miniprepped plasmid
*I have already PCR-magnified iGEM10_002 and iGEM10_003 with the appropriate Gibson oligos I designed
*incubated for 1hr at 37
''Procedure''<br> Well contents:
*zymo cleaned FP digests (eluted with 20uL), small fragment zymo cleaned Bca1152 (
*50μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA
====Ligation====
*recipe:
**10uL vector digest (Bjh2251 or Bjh2251)
**7.5uL Bca1152
**2uL Ligase
**2uL T4 buffer +ATP
*let sit on counter for 30 min


{| {{table}}
====Transformation====
| align="center" style="background:#f0f0f0;"|'''Wells'''
*cell mixture: 100uL Jtk030, 15uL KCM
| align="center" style="background:#f0f0f0;"|'''Part'''
*50uL of cell mixture added to each ligation mixture
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
*let sit on ice for a few minutes
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
*heat shocked at 42*C for 90 sec
|-
*added 100uL 2yt
| 1||iGEM10_001 a||ca998||igemTen002
*rescued for 45 min
|-
*plated both on CA
| 2||iGEM10_001 b||ca998||igemTen002
|-
| 3||iGEM10_001 c||ca998||igemTen002
|-
| 4||<piggybac!||igemTen001||igemTen004
|-
| 5||Self Lysis Device(Bjh2294)||igemTen007||G00101
|-
| 6||1601AC plasmid ||igemTen009||igemTen010
|}
*Run on PCR program 4K55 <br>
'''Gel Results''' <br>[[Image: ConorGel5.jpg||400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_001 a||ca998||igemTen002||1360|| No, not visible
|-
| 3||iGEM10_001 b||ca998||igemTen002||1360||No, not visible
|-
| 5||iGEM10_001 c||ca998||igemTen002||1360||No, not visible
|-
| 7||<piggybac!||igemTen001||igemTen004||1820||No, not visible
|-
| 9||Self Lysis Device(Bjh2294)||igemTen007||G00101||2540||No, not visible
|-
| 11||1601AC plasmid||igemTen009||igemTen010||3220||No, not visible
|}
==== 2nd Attempt at PCR: Using Expand Polymerase====
*33μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Wells'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
|-
| 1||iGEM10_001 a||ca998||igemTen002
|-
| 2||iGEM10_001 b||ca998||igemTen002
|-
| 3||iGEM10_001 c||ca998||igemTen002
|-
| 4||<piggybac!||igemTen001||igemTen004
|-
| 5||Self Lysis Device(Bjh2294)||igemTen007||G00101
|-
| 6||1601AC plasmid ||igemTen009||igemTen010
|}
*Ran on 5K55 and left on thermocycler overnight (see gel results on tomorrow's notebook entry)


==[[User:Conor McClune|Conor McClune]] 14:11, 11 June 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 14:40, 12 August 2010 (EDT)==
===Oligo and Composite Parts Test PCR #2 (continued from yesterday)===
===PCR magnification of Pcon part Bca1152===
15uL of PCR product run on gel:<br>
*template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
[[image:ConorGel2.jpg|400px]]
*recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
{| {{table}}
*run on 2K55
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Matches Gel Results?'''
|-
| 1||iGEM10_002d||igemTen003||G00101||500||yes
|-
| 2||iGEM10_002d||ca998||igemTen006||470||yes
|-
| 3||iGEM10_002d||ca998||G00101||570||yes
|-
| 4||iGEM10_003b||igemTen005||G00101||1190||yes
|-
| 5||iGEM10_003b||ca998||igemTen008||1160||yes
|-
| 6||iGEM10_003b||ca998||G00101||1260||yes
|-
| 7||iGEM10_003d||igemTen005||G00101||1190||yes
|-
| 8||iGEM10_003d||ca998||igemTen008||1160||yes
|-
| 9||iGEM10_003d||ca998||G00101||1260||yes
|}
<br>
<br>
'''Gel Verification'''
*expected size:1079
[[image:ConorGel98.jpg|100px]]<br>
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
[[image:ConorGel99.jpg|200px]]
*no visible bands
==[[User:Conor McClune|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
<br>
<br>
'''Gel Verification'''
*expected size:1079
*row 1 : 10uL phusion product
*row 2 : 10uL expand product


===Oligo and Composite Parts Test PCR #3===
[[image:ConorGel97.jpg|100px]]
''Procedure''
*both products are the correct size, though yield is weak
Well contents:
*Phusion product looks best
*50μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA
 
===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
 
==[[User:Conor McClune|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
 
==[[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;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_20 (atc)'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_20 (no atc)'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_013 (atc)'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_013 (no atc)'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_131 (atc)'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_131 (no atc)'''
|-
|-
| 1||Taq||iGEM10_002d||igemTen003||igemTen006
| Transposase Buffer||32||5||7||8||very high density||high density 
|-
|-
| 2||Phusion||iGEM10_002d||igemTen003||igemTen006
| ASW||70||135||35||49||high density||higher density
|-
|-
| 3||Taq||iGEM10_003b||igemTen005||igemTen008
| LB||490||146||119||105||high density (almost lawn)||high density (almost lawn)
|-
|-
| 4||Phusion||iGEM10_003b||igemTen005||igemTen008
| TB||234||158||73||122||0||high density (almost lawn)
|}
|}
*Wells run on 2K55 on thermocycler.
 
''Gel Results''<br>
==[[User:Conor McClune|Conor McClune]] 14:31, 5 August 2010 (EDT)==
[[image:ConorGel3.jpg|400px]]
===Transposase Assay Results===
*possible DNA spilling from lane 4 to lanes 3&5, thus I repeated the gel run to avoid a false positive in lane 5 <br>
No growth on any plates
[[image:ConorGel4.jpg|400px]]
===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;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Name'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Target Part'''
| 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;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Matches Gel Results?'''
|-
| 1||Taq||iGEM10_002d||igemTen003||igemTen006||400||yes, strong band
|-
| 2||Phusion||iGEM10_002d||igemTen003||igemTen006||400||yes, weaker band
|-
|-
| 3||Taq||iGEM10_003b||igemTen005||igemTen008||1100||yes, strong band
| 1||sbb10||KA||<SB100x!||ca998||igemTen048
|-
|-
| 4||Phusion||iGEM10_003b||igemTen005||igemTen008||1100||yes, weaker band
| 2||sbb04||KA||<piggyBac!||ca998||igemTen050
|-
|-
| 5||n/a||pB6||||||40,000||possible band at 40kb, but very low conc.
| 3||iGEM10_055||KA||<tn5>{<NIS!}||ca998||igemTen051
|}
|}


===Gel Purification of iGEM10_002 and iGEM10_003===
==[[User:Conor McClune|Conor McClune]] 14:30, 4 August 2010 (EDT)==
* cut out Phusion transcribed bands:
===Transposase Assay: Testing Sleeping Beauty and Tn5 in NEB2+MgCl2, varying [DNA] and activity time===
**band at 400 in lane 2
**band at 1100 in lane 4
*followed [http://openwetware.org/wiki/Template:SBB-Protocols_Zymo3 this protocol] and placed the purified PCR products of iGEM10_002 and iGEM10_003 in the working box


===PDD Part Preparation===
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.
====Eco/Bam Transfers====
*Eco/Bam transfered 1601CA Bjh 2333 L, 1601CA Bjh2348 L,1601CA Bjh2349, and 1601CA Bjh 2331 in pBca1601 AK( we got this from shelley, and had to dilute it by half). We did this because BAC is CAM resistant, so if both parts are Cam resistant, it is possible to lose BAC, which is only single, and not be able to tell. So in the end, we will end up plating our stuff on A/K/C/Spec plates.


1601 AK or KA into MG1655 or MC1061
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]] 13:23, 10 June 2010 (EDT)==
Added 1uL of atc and 10uL of arabinose to each mL of cells at 1:05pm. Put in 37deg shaker.
'''Negative Controls from Yesterday's Competent Cells'''
*plates: Amp,Kan,Cam,Spec
*positions: 1=pBca1256-Bjh2343, 2=pBca1256-Bjh2344, 3=pBca1256-Bjh1934, 4=MG1655
[[Image:NegControlAmp.jpg|200px]]
[[Image:NegControlKan.jpg|200px]]
[[Image:NegControlCam.jpg|200px]]
[[Image:NegControlSpec.jpg|200px]]<br> <br>


'''Plating yesterdays BAC tranformations failed'''<br>
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).
[[Image:BAC2343.jpg|200px]]
[[Image:BAC2344&1934.jpg|200px]]<br>
''Retransformation''
* Transforming pB6 BAC into pBca1256-Bjh2343, pBca1256-Bjh2344 and pBca1256-Bjh1934
*adjustments to [http://openwetware.org/wiki/Template:SBB-Protocols_Micro1 procedure]:
**using only 15uL KCN for 98uL competent cells
**NOT diluting DNA by 10 before adding to cell solution
**For each strain, we split cell/KCN solution into two tubes of 57uL: we added 1μL DNA to one tube and 3 μL to the other tube
**after rescue, entire ~115uL of each solution was plated onto its own plate
<br> <br>


'''Oligo and Composite Parts Test PCR'''
Somehow, we ran out of MP, so we're only adding up to 10uL of DNA.
Composite Parts ready to test:
*iGEM10_002d
**oligos:
***igemTen003
***igemTen006
*iGEM10_003b
**oligos:
***igemTen005
***igemTen008
*iGEM10_003d
**oligos:
***igemTen005
***igemTen008
Procedure:
*Dilute each oligo by ten (5ul oligo in 45ul MG H20)
*Combine in PCR tubes:
**50uL Phusion MM
**1uL oligo 1
**1uL oligo 2
**0.5uL miniprepped DNA
*program 2K55 started at 12:10 (should be done at 2:30)
Gel Results:
*no visible DNA in any of the lanes
[[Image:ConorGel1.jpg|200px]] <br> <br>
'''Oligo and Composite Parts Test PCR #2'''
*Parts tested:
**iGEM10_002d
***oligos:
****igemTen003(fwd)
****igemTen006(rev)
***iGEM10_003b
***oligos:
****igemTen005(fwd)
****igemTen008(rev)
***iGEM10_003d
***oligos:
****igemTen005(fwd)
****igemTen008(rev)
*for each part, testing:
**fwd part oligo + standard reverse plasmid oligo(G00101)
**rev part oligo + standard forward plasmid oligo(ca998)
**standard reverse plasmid oligo(G00101 + standard forward plasmid oligo(ca998)


Lanes:
Time points: <br>
#iGEM10_002d+igemTen003+G00101
.5hr- 2:10pm <br>
#iGEM10_002d+ca998+igemTen006
1hr- 2:40pm <br>
#iGEM10_002d+ca998+G00101
1.5hr- 3:10pm <br>
#iGEM10_003b+igemTen005+G00101
2hr- 3:40pm
#iGEM10_003b+ca998+igemTen008
#iGEM10_003b+ca998+G00101
#iGEM10_003d+igemTen005+G00101
#iGEM10_003d+ca998+igemTen008
#iGEM10_003d+ca998+G00101


PCR run and products kept at 16*C in the thermocycler overnight
Transform into TG1 cells. We mixed 11 tubes of TG1 cells together in a Falcon tube.


==[[User:Conor McClune|Conor McClune]] 15:08, 9 June 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 16:53, 3 August 2010 (EDT)==
'''Created -80 Stocks'''
===Assembly of iGEM10_007===
*used [http://openwetware.org/wiki/-80_Glycerol_Stocks this protocol] to make -80 stocks for
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.
**pBca1256-Bjh1934 in MG1655
**pB6 in DH5-alpha
**MG1655
**Bjh2343 in MG1655
**Bjh2344 in MG1655
*scanned and logged these stocks as the first iGEM -80 stocks (created a stock box for -80)


'''Made Competent Cells'''
===Analytical PCR of iGEM10_131===
*for pBca1256-Bjh1934 in MG1655,Bjh2343 in MG1655 and Bjh2344 in MG1655:
Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
**added 500μL of overnight-grown culture to 50mL Spec LB in an Erlenmeyer flask
*run on 4K45
*for MG1655:
{| {{table}}
**added 1mL of overnight-grown culture to 50mL LB (no AB) in an Erlenmeyer flask (because the solution had been diluted 1:2 with 50% glycerol)
| align="center" style="background:#f0f0f0;"|'''Well'''
*both flasks put on shaker in lab at 12:00
| align="center" style="background:#f0f0f0;"|'''Part'''
| 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_103||ca56||igemTen004||2045||yes
|-
| 2||iGEM10_103||ca56||igemTen006||2411||yes
|-
| 3||iGEM10_103||ca56||igemTen008||3475||no
|}
[[image:ConorGel96.jpg|400px]]


*2:00pm
===Preparation of Bacterial Stock Choano Food===
** pBca1256-Bjh1934,Bjh2343 and Bjh2344 (all in MG1655) have all grown up well, but not MG1655 (probably because of the glycerol)
*Grew up 400mL MC1061 overnight on 37*C shaker
*** MG1655 was place back in the incubator
*split into two centrifuge bottles and centrifuged at 4200rpm for 14 minutes
*** pBca1256-Bjh1934,Bjh2343 and Bjh2344 solutions were chilled for ten minutes before being transferred to 50mL Falcon tubes and centrifuged for 13 minutes on 4100rpm.
*poured off supernatant
****poured off the supernatant
*washed each pellet with 10mL artificial sea water (ASW)
****added 2.5ml TSS solution to each of the three vials
*poured off ASW
****made 98uL aliquots into 25 chilled eppendorf tubes for storage (25 tubes for each of pBca1256-Bjh1934,Bjh2343 and Bjh2344)
*added 10mL ASW to each bottle and resuspended pellets
*Transformed BAC into 1, 2, 3.
*centrifuged at 4200rpm for 14 minutes
*Plated on Cam, Spec plates.
*poured off supernatant
*resuspended pellets
*combined the contents of the two bottles
*created 2uL aliquots
*


*3:30
==[[User:Conor McClune|Conor McClune]] 14:39, 2 August 2010 (EDT)==
** removed MG1655  from incubator and conducted the procedure above (chilling, centrifuging, resuspending in TSS and aliquoting)
** 100 tubes labeled "1" (for Bjh2343), "2" ( Bjh2344), "3"(pBca1256-Bjh1934), "4"(MG1655 )
** Drops of the remaining solutions in the Falcon tubes were dropped on plates of Amp, Can, Spec, and Chlor as a negative control


==[[User:Conor McClune|Conor McClune]] 13:52, 8 June 2010 (EDT)==
===Miniprep of iGEM10_103===
*The five plates from yesterday (shown below) grew up well. We picked two colonies from the following:
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.
**2343
**2344
**1256-1934
**MG1655
**pB6
*The first four of these will mixed with glycerol to be used as competent cells
*Picked Transformed cells from Stage 1 of Assembly of iGEM10_007
**plated on ACK plate to check for cotransformation
**incubated in shaker
**ran colony PCR
====Colony PCR====
Selected four colonies from each plate. Dipped the pipette tip in PCR tube with 40uL of MasterMix and a well containing 3mL of LB with the appropriate antibiotics, before spreading the pipette tip across a plate with all three antibiotics (to check for cotransformation).


iGEM10_001 (part 8 next to 23 in a CK vector): Expected band length: about 1500bp <br>
====Restriction Mapping====
iGEM10_002 (part 11 next to 6 in AC vector): Expected band length: about 550bp <br>
*Digested 4uL of each miniprep with Xho/Bgl. Incubated from 11:30-12:15. Froze from 12:15-1:15.
iGEM10_003 (part 20 next to 5 in CK vector): Expected band length: about 1250bp <br>
*expected fragments: 6995 (desired fragment), 1607 (vector fragment)
[[image:ConorGel93.jpg|400px]]
*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.


[[Image:6-8-10.jpg]]


Colony PCR<br>
====Analytical PCR====
Lane 1 iGEM10_003a (should be 1250bp) <br>
Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
Lane 2 iGEM10_003b (should be 1250bp) <br>
*run on 4K45
Lane 3 iGEM10_003c (should be 1250bp) <br>
{| {{table}}
4 iGEM10_003d (should be 1250bp) <br>
| align="center" style="background:#f0f0f0;"|'''Well'''
5 iGEM10_001a (should be 1500bp) <br>
| align="center" style="background:#f0f0f0;"|'''Part'''
6 iGEM10_001b (should be 1500bp)<br>
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
7 iGEM10_001c (should be 1500bp)<br>
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
8 iGEM10_001d (should be 1500bp)<br>
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
9 iGEM10_002a (should be 550bp)<br>
| align="center" style="background:#f0f0f0;"|'''Acceptable Clones'''
10 iGEM10_002b (should be 550bp)<br>
|-
11 iGEM10_002c (should be 550bp)<br>
| 1||iGEM10_103||ca998||igemTen004||1859||1-3,5,6
12 iGEM10_002d (should be 550bp)<br>
|-
 
| 2||iGEM10_103||ca998||igemTen006||2225||1-3,5,6
We will choose the following samples to miniprep tomorrow:<br>
|-
iGEM10_001c,d <br>
| 3||iGEM10_103||ca998||igemTen008||3289||none
iGEM10_002c,d <br>
|}
iGEM10_003a,b <br>
 
*Moved the sequences from BioE 140L from Clotho to the Data Sheet on Google Docs
*Created oligos for a Gibson PCR for the assembly of iGEM10_007
**logged as the first oligos on Google Docs
**ordered by Tim


==[[User:Conor McClune|Conor McClune]] 14:38, 7 June 2010 (EDT)==
'''Gel Pictures'''<Br>
[[Image:jin_Notes1.JPG|200px]]<br>
*ca998 + igemTen004
*(with Tahoura)
[[image:ConorGel94.jpg|200px]]
*Transformed (spec)1256-2343 and (spec)1256-2344 into MG1655. Used minipreps of spec1256-2343 and spec1256-2344 from box and diluted 10x. Mixed 50ul MG1655 with 17.5ul KCN and added 1ul diluted DNA. Incubated for 1hr and plated on Spec. Left in incubator to grow <br>
*ca998 + igemTen006 (lanes 1-6) and ca998 + igemTen008 (lanes 7-12)
*Plated pB6 (BAC), place in incubator. Needs to be picked and miniprepped tomorrow.<br>
[[image:ConorGel95.jpg|200px]]
*Transformed pBca1256-Bjh1934 into MG1655. Used procedure from (1)
*Attempted to enter parts into clotho --> would not save


===First step of Manual Assembly===
'''Results'''<br> Either a part is missing around the 3'TerminalRepeat, or the pcr is bad. I will repeat it tomorrow


===Assembly of iGEM10_007===
====Digestion====
====Digestion====
Lefty MM added to 9+8, 7+11, 9+20<br>
*I used a stored, previously digested version of iGEM10_001
Right MM added to 7+23, 8+6, 7+5 <br>
*I used the above Xho/Bgl digestion of iGEM10_103 (aka iGEM10_006)
They should be done incubating at 3:48pm.<br>


====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


'''Lefty MasterMix''' (for 4uL miniprep DNA)
===Transformation===
*4uL water
*cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
*1uL of NEB2
*added 70uL of this cell mixture to the ligation mixture
*0.5uL XhoI
*heat shocked for 90sec
*0.5uL BamH1
*added 100uL 2YT and rescued for 1 hr


'''Righty MasterMix''' (for 4uL miniprep DNA)
===Transformed iGEM10_013 #5===
*4uL water
*transformed into JTK030 cells for use in a transposase assay
*1uL of NEB2
*cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
*0.5uL XhoI
*added 70uL of this cell mixture to the ligation mixture
*0.5uL BglII
*heat shocked for 90sec
*added 100uL 2YT and rescued for 1 hr


*note: righty digestions had different volumes, which seemed suspicious, so the digestion was repeated with 1uL of each enzyme for 30 min.
===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


====Zymo====
==[[User:Conor McClune|Conor McClune]] 23:44, 1 August 2010 (EDT)==
Means of getting rid of restriction enzymes. Didn't heat kill because BamH1 isn't denatured by heat killing. Eluted with 10uL of H2O. Products are labeled 7+11 L, 9+8 L, 9+20 L.
===Picking colonies for iGEM10_103 (iGEM10_006)===
 
*Picked 6 colonies - grew up in KA LB
====Ligation:====
*Colony PCR
Followed [http://openwetware.org/wiki/Template:SBB-Protocols_Enz4 this protocol] with the following amendments: <br>
**primers: igemTen004 and ca56
Mastermix
*6.5uL ddH2O
*1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
*0.5uL T4 DNA Ligase
DNA
*3uL Lefty vector
*3uL Right vector
Added DNA to MM at 5:12pm. Will incubate on bench until 5:42pm.
 
===Transformation===
*70uL  of each ligation added to cells
**7+11/8+6 in righty
** 9+8/7+23 and 9+20/7+5 in lefty

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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