Berk2010-Conor (June)

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Conor McClune 20:42, 30 June 2010 (EDT)

Tn5 Transposase Assay

Testing to see whether the Transposase if functional.

Specifically, we varied atc concentration and the time allowed for transposase activity.

Inoculated cells drived from another colony off the iGEM10_020A plate, which didn't undergo co-transformation. We can use these for tests tomorrow.

  • Added 10uL of 20% Arabinose (100X) to 1mL of lefty pir+ cells, transformed with iGEM10_020A (no co-transformation occured) at 1:30pm.
  • At 2:30pm, will add varying amounts of atc to cells.
    • 1uL of 1000X atc diluted 1:10
    • 1uL of 1000x atc
    • 3uL of 1000x atc
  • At 3:30pm, will add 1uL of pBca9145-Bca1144DNA, and allow the transposase to do it's thing for varying amounts of time.
    • 3:45pm (15min):Remove 250uL of lysate from each tube and spin down and zymo the supernatant.
    • 4:00pm (30min): See above
    • 4:15pm (45min): See above
    • 4:30pm (1hour): See above (note: less than 250uL was left at this point)
  • Transformed each of the 12 zymo purified DNAs into 70uL wild-type mc1061 cells
  • incubated for 1 hours
  • plated on Amp/Gen plates to grow up overnight

Results

  • no colonies on any of the 12 plates


Note: Talked to Terry, and he said one thing we definitely need to vary is the amount of time we give for Transposase expression.

Potential Problem: Used 2YT-Amp for the tubes colored with green sharpie. Hopefully this won't be a problem, since it's sorta the same thing as immediately plating on Amp.

Conor McClune 14:36, 29 June 2010 (EDT)

Gibson Assembly of iGEM10_007 and iGEM10_013

Restriction Mapping

In order to save time, I also am running a digest of iGEM10_007 for convenience, and of Bth030 for comparison (because it is about 6kb in length).
Well contents:

well Contents Enzymes Expected Size
1iGEM10_013 #14Eco/Bam6204
2iGEM10_013 #16Eco/Bam6204
3iGEM10_007 #11Eco/Bam7060
4iGEM10_007 #13Eco/Bam7060
5Bth030Eco/Bam5850

Analytical PCR

Well Sample Part Checked fwd oligo rev oligo expected size
1iGEM10_013 #14iGEM10_001ca998igemTen0021435
2iGEM10_013 #14B10sbb10igemTen034igemTen0361026
3iGEM10_013 #14iGEM10_009igemTen035igemTen018279
4iGEM10_013 #14iGEM10_008igemTen017igemTen0201057
5iGEM10_013 #14Bjh2294 w/ fwd oligo 19igemTen019G001012607
6iGEM10_013 #16iGEM10_001ca998igemTen0021435
7iGEM10_013 #16B10sbb10igemTen034igemTen0361026
8iGEM10_013 #16iGEM10_009igemTen035igemTen018279
9iGEM10_013 #16iGEM10_008igemTen017igemTen0201057
10iGEM10_013 #16Bjh2294 w/ fwd oligo 19igemTen019G001012607

Repicking and Colony PCR

  • picked 8 colonies each of iGEM10_007 and iGEM10_013
  • ran two colony PCRs for each colony using the following oligos:
    • for 007
      • ca56 + igemTEN4
      • igemTEN3+igemTEN8
    • for 013
      • ca56+igemTEN36
      • igemTEN35 + igemTEN20

Gel Results

  • lane organization:
    • lanes 1-16 are eight 007 colonies, odd lanes have primers ca56 + igemTEN4, even lanes have primers igemTEN3+igemTEN8
    • lanes 1-16 are eight 013 colonies, odd lanes have primers ca56 + igemTEN36, even lanes have primers igemTEN35+igemTEN20


Results: There are no distinct bands in any lanes. At this point, I think it best to do another round of 2AB assembly before attempting gibson again for parts iGEM10_007 and iGEM10_013.

Conor McClune 18:13, 28 June 2010 (EDT)

Choano Heat/NH4 Assays

  • on Friday (3 days ago), I split choanos 1:8 into choano media under the following conditions:
    • 25*C(control)
    • 37*C incubator
    • 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, however, here are the counts I did get from PI staining:
    • control: 1/4 dead
    • 1mM NH4: 12/38 dead
    • 37*C incubator:7/17 dead
  • I got a fresh culture of MbFb from the king lab so I can redo this experiment starting tomorrow

iGEM10_007

Analytical PCR

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

  • run on 4K45
Well Sample Part Checked fwd oligo rev oligo expected size
1iGEM10_007 #11iGEM10_001ca998igemTen0021435
2iGEM10_007 #11 (B10sbb04)igemTen001igemTen0041788
3iGEM10_007 #11iGEM10_002igemTen003igemTen006366
4iGEM10_007 #11iGEM10_003igemTen005igemTen0081064
5iGEM10_007 #11Self Lysis Device (Bjh2294)igemTen007G001012607
6iGEM10_007 #13iGEM10_001ca998igemTen0021435
7iGEM10_007 #13 (B10sbb04)igemTen001igemTen0041788
8iGEM10_007 #13iGEM10_002igemTen003igemTen006366
9iGEM10_007 #13iGEM10_003igemTen005igemTen0081064
10iGEM10_007 #13Self Lysis Device (Bjh2294)igemTen007G001012607

iGEM10_013

Analytical PCR

Well contents: 33uL Taq MM, 1uL each primer, .5 uL template DNA

Well Sample Part Checked fwd oligo rev oligo expected size
1iGEM10_013 #14iGEM10_001ca998igemTen0021435
2iGEM10_013 #14B10sbb10igemTen034igemTen0361026
3iGEM10_013 #14iGEM10_009igemTen035igemTen018279
4iGEM10_013 #14iGEM10_008igemTen017igemTen0201057
5iGEM10_013 #16iGEM10_001ca998igemTen0021435
6iGEM10_013 #16B10sbb10igemTen034igemTen0361026
7iGEM10_013 #16iGEM10_009igemTen035igemTen018279
8iGEM10_013 #16iGEM10_008igemTen017igemTen0201057
9iGEM10_013 #14Bjh2294 w/ fwd oligo 19igemTen019G001012607
10iGEM10_013 #16Bjh2294 w/ fwd oligo 19igemTen019G001012607
  • I placed this on the plate and selected the program, but failed to hit start, so the wells sat overnight at room temp.
  • I restarted the program in the morning, but it is doubtful whether it will yield good results.

Conor McClune 19:19, 27 June 2010 (EDT)

iGEM10_013

Picking colonies/Colony PCR

  • picked 16 colonies
  • oligos used for col PCR: ca56 and igemTen020
    • expected band size = 2619

Gel Results
(These are the same gels, but run for different lengths)

Results

  • no bands in any of the chosen colonies, however it seems from the short run on the gel that at least one of the primers was binding to to colonies #14 and 16, as there is a large amount of small fragments of DNA

iGEM10_020

Miniprep

Friday's col PCR showed that all the colonies selected for the second col PCR had iGEM10_001 transferred into it. The last 4 lanes appeared to have the strongest bands, so I will choose those colonies to miniprep. I will call colonies represented by lanes 13-16 iGEM10_020 a-d, respectively.

Conor McClune 14:53, 25 June 2010 (EDT)

iGEM10_020

Bacterial lawns grew from both of the transformations from yesterday. Since there is no antibiotic for the correctly Eco/Bam/Bgl transfered product, I will have to do a number of colony PCRs to find one that took up the iGEM10_001 part.

Colony PCR

  • oligos: ca56 (fwd) and igemTen22(rev)
  • picked 12 colonies from igem10_020 #15 and 4 colonies from igem10_020 #12

Gel Results

  • igem10_020 #15

  • igem10_020 #12


Results: no insertion in any of the chosen colonies

  • I may have forgotten to add DNTPs, which would explain why there are no bands



I repicked 32 more colonies from igem10_020 #15 and ran a col PCR. Gel Results

  • igem10_020 #15 (expected band size: 1803)

  • it appears each of the colonies have a plasmid that has iGEM10_001

iGEM10_013

Gibson Assembly

  • DNA contents of assembly:
Part Size Band Brightness Weight Volume added (uL)
iGEM10_0011335bright0.50.714285714
B10sbb101026bright0.50.714285714
iGEM10_009279dim0.50.714285714
iGEM10_0081057bright0.50.714285714
Bjh2294 w/ fwd oligo 192507bright0.50.714285714
p1601AC (vector)3183medium bright11.428571429
total3.55
  • mixed with 15uL Gibson MM
  • incubated at 50*C for 60 minutes
  • transformed in to mc1061 cells

Conor McClune 14:25, 24 June 2010 (EDT)

iGEM10_020

EcoR1/BamHI, EcoR1/Bglii Transfer

Digestion

  • the following wells were incubated at 37 for 30 min:
Well Part Buffer Enzyme1 Enzyme2 Water
14uL iGem10_020 Gibson Colony #12 1uL NEB31uL Eco1uL Bgl4ul
24uL iGem10_020 Gibson Colony #15 1uL NEB31uL Eco1uL Bgl4ul
38uL iGem10_001 c1uL NEB22uL Eco2uL Bam8ul


I zymo purified the contents of the three wells, and spun it down into an equal volume of water as each digestion mixture (10,10,20uL)

Ligation I incubated the following on my desk for 30 min:

Well Part1 Part2 Buffer Enzyme1 Water
110uL iGem10_020 Gibson Colony #12 10uL iGem10_001 c10uL Ligase Buffer5uL T4 Ligase65ul
210uL iGem10_020 Gibson Colony #15 10uL iGem10_001 c10uL Ligase Buffer5uL T4 Ligase65ul

iGEM10_007

  • sequencing company reported that oligo ca56 did not work with iGEM10_007, as it should have.
  • I ran a diagnostic PCR to see what was wrong:
Well Part fwd oligo rev oligo Expected Size
1iGEM10_007 #3ca56igemTen002256
2iGEM10_007 #3ca56igemTen0042044
3iGEM10_007 #3igemTen003igemTen0081462
4iGEM10_007 #3igemTen007G001012635
5iGEM10_007 #7ca56igemTen002256
6iGEM10_007 #7ca56igemTen0042044
7iGEM10_007 #7igemTen003igemTen0081462
8iGEM10_007 #7igemTen007G001012635

Gel Results

iGEM10_013

  • was able to continue parts preparation for Gibson assembly because my last primers arrived

Parts PCR
Well contents: 33uL polymerase,1uL each oligo, .5uL template DNA

  • run on program 2K45
Lane Part Fwd Oligo Rev Oligo Polymerase Predicted Size
1 B10sbb10igemTen034igemTen036Expand1050
2iGEM10_009igemTen035igemTen018Expand300
3 B10sbb10igemTen034igemTen036Phusion1050
4iGEM10_009igemTen035igemTen018Phusion300

'Gel Results'

Conor McClune 19:50, 23 June 2010 (EDT)

Manual Assembly of iGEM10_004 and iGEM10_005

  • no growth on either of the plates, again
  • something must be wrong with ligation or digestion
  • however, this was merely a backup for the Gibson Assembly of iGEM10_007, which seems to be working, so there is no need to troubleshoot

Gibson Assembly of iGEM10_007

  • sent miniprepped DNA in for sequencing
  • I chose colony #3, because the restriction digest band was the brightest

Gibson Assembly of iGEM10_020

  • miniprepped colonies 1,3,4,11,12,14,15
  • digested with Eco/Bam for restriction mapping
  • Results:

Lane 8 (colony #15) is the only one that displays the correct bands

Conor McClune 15:34, 22 June 2010 (EDT)

Manual Assembly of iGEM10_004 and iGEM10_005

  • no growth on plates from yesterday, probably because I used only 1uL of ligation mixture for each transformation
  • luckily, I kept the ligation mixture, so I redid the transformations, using the remaining ligation mixture for each of the transformations

Gibson Assembly of iGEM10_020

  • plenty of colonies on both plates
  • picked 8 colonies from each plate, and set up a colony PCR with oligos ca998 and igemTen22
  • expected magnified fragment length is 1617
  • colony PCR results:
    • plate 1(colonies 1-8):

    • plate 2(colonies 9-16):

  • These colonies look good: 1,3,4,11,12,14,15

Gibson Assembly of iGEM10_007 (2nd Attempt)

  • miniprepped growth from yesterdays promising colonies (colonies 2,3,5,7,10,11,13,15)
    • created 50uL DNA
  • Eco/Bam digested 4uL of each for restriction mapping
    • mapping results (expected sizes are 7060 and 3200) :

  • Results:
    • 7060 band is faint but definitely present in each lane
    • 3200band is probably present, but too faint to see, considering it is shorter
    • these look good and ready for sequencing confirmation

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 (colonies 1-8):

  • expected band size: 9000
  • gel picture:

  • Results:
    • colonies 1,4,6,8 are too short
    • colonies 2,3,5,7 show nothing, which is hopeful, since Taq generally cannot copy much larger than 5kb

With iGEM primers 1 and 6 (colonies 9-16):

  • expected band size: 2186
  • gel picture:

  • Results:
    • no strong bands
    • numerous faint bands in lanes 2,3,5,7 (colonies 10,11,13,15) -- This suggests that something is being read off the primers
      • These colonies are promising because the primers are from within the part

Gibson Assembly of iGEM10_020

  • DNA contents:
Part PCR Purified Size Band Brightness Weight Volume added (uL)
iGEM10_017yes1546bright0.50.833333333
iGEM10_016yes858medium dim11.666666667
Bjh2294 with fwd oligo 23yes2507bright0.50.833333333
p1601AC (vector)yes3183medium bright11.666666667
total:35
  • added 15uL Gibson MM
  • incubated at 50*C for 70 minutes
  • transformed the entire mixture into mc1061 cells
  • plated cells on two CA LB plates incubated overnight

Manual Assembly of iGEM10_004 and iGEM10_005

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

Conor McClune 19:16, 20 June 2010 (EDT)

Gibson Chemistry

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

  • 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:
Part PCR Purified Size Band Brightness Weight Volume added (uL)
iGEM10_001yes1335 bright0.250.3125
(B10sbb04)yes1788 bright0.250.3125
iGEM10_002yes366dim0.50.625
iGEM10_003yes1064dim11.25
Self Lysis Device (Bjh2294)yes2507medium bright11.25
pBjh1601AC (vector)yes3183medium bright11.25
total45
  • 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)

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

  • 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



DpnI Digestion of Bjh1601 AC

  • 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



PCR of Bjh2294 with fwd oligos 19 and 23"
Well Contents: 50uL phusion MM, 0.5uL DNA template, 1uL each oligo

Well Part Polymerase Fwd Oligo Rev Oligo Thermocycler Prgm
1Bjh2294PhusionigemTen19G001014K55
2Bjh2294PhusionigemTen23G001014K55
3Bjh2294PhusionigemTen19G001014K45
4Bjh2294PhusionigemTen23G001014K45

Gel Results (the lane numbers correspond the the well numbers above)

  • bands in lane 3 and 4 are strong and match the expected 2507bp
    • I gel purified these bands

Gibson Assembly of iGEM10_007

Well contents:

' Volume added (uL)
iGEM10_0010.833
<piggybac!(B10sbb04)0.833
iGEM10_0020.833
iGEM10_0030.833
Self Lysis Device (Bjh2294)0.833
p1601AC (vector)0.833
Gibson MM20
  • 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)

Conor McClune 13:54, 17 June 2010 (EDT)

Gibson Chemistry

Parts Preparation

DpnI Digestion
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


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

  • Well contents: 33uL Phusion MM, 1uL each oligo (ss13r and igemTen009), .5uL template DNA
  • run on program 4K55

  • 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


PCR of sbb10

  • Continued attempts to PCR sbb10
  • double checked oligos on ApE
  • created new 1:10 dilutions of oligos igemTen 14 and igemTen16
  • 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
Lane Part Polymerase Fwd Oligo Rev Oligo
1sbb10PhusionigemTen14igemTen16
2sbb11ExpandigemTen14igemTen16
3sbb12PhusionigemTen14igemTen16
4sbb13ExpandigemTen14igemTen16

Wells 1,2 run on program COL2K
Wells 3,4 run on program 2K45

  • 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

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

Well Part Polymerase Fwd Oligo Rev Oligo Size Expected Acceptable?
1B10sbb10ExpandigemTen014igemTen016260Very faint
2iGEM10_009 AExpandigemTen015igemTen018310 Yes
3iGEM10_008d ExpandigemTen017igemTen0201090 Yes
4iGEM10_017 AExpandca998igemTen0221650 Yes
5iGEM10_016aExpandigemTen021igemTen024890 Yes
6pB6 (BAC)ExpandigemTen025igemTen0261000Yes
  • 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)
Well Part Polymerase Fwd Oligo Rev Oligo
1B10sbb10PhusionigemTen014igemTen016
2B10sbb10ExpandigemTen014igemTen016
3Bjh2294PhusionigemTen007G00101
4Bjh2294ExpandigemTen007G00101
5Bjh2294 (PCR Product)PhusionigemTen007G00101
    • Strip 2: run on 4K55(max temp=55*C)
Well Part Polymerase Fwd Oligo Rev Oligo Size Expected
1B10sbb10PhusionigemTen014igemTen016260
2pBjh1601PhusionigemTen009ss13r3183
3pBjh1601ExpandigemTen009ss13r3183

Gel Results

Lane Part Polymerase Fwd Oligo Rev Oligo Size Expected Acceptable?
1B10sbb10PhusionigemTen014igemTen016260 no
2B10sbb10ExpandigemTen014igemTen016260 no
3Bjh2294PhusionigemTen007G001012540 Probably just template DNA
4Bjh2294ExpandigemTen007G001012540no
5Bjh2294 (PCR Product)PhusionigemTen007G001012540 yes
6-----
7B10sbb10PhusionigemTen014igemTen016260 no
8pBjh1601PhusionigemTen009ss13r3183 yes
9pBjh1601ExpandigemTen009ss13r3183no
  • none of the B10sbb10 PCR attempts worked
  • I gel purified the desired bands in lanes 5 and 8: Bjh2294 (PCR Product) and pBjh1601

Conor McClune 14:00, 15 June 2010 (EDT)

Gibson Chemistry

Parts PCR

Gel Results from yesterday's Expand PCR

  • note: it appears some of well 6 (p1601ac) has evaporated. Perhaps the lid was not closed completely.

  • gel looked messy, so I ran it again:

Lane Part Fwd oligo Rev oligo Predicted Size Acceptable?
1iGEM10_001 aca998igemTen0021360 Yes
3iGEM10_001 bca998igemTen0021360Yes
5iGEM10_001 cca998igemTen0021360Yes
7<piggybac! (sbb04)igemTen001igemTen0041820Yes
9Self Lysis Device (Bjh2294)igemTen007G001012540Very Faint
111601AC plasmidigemTen009igemTen0103220No, 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

Well Part Polymerase Fwd oligo Rev oligo
1Self Lysis Device(Bjh2294)PhusionigemTen007G00101
2Self Lysis Device(Bjh2294)ExpandigemTen007G00101
3Self Lysis Device(Bjh2294)TaqigemTen007G00101
41601AC plasmidPhusionigemTen009G00101
51601AC plasmidExpandigemTen009G00101
61601AC plasmidTaqigemTen009G00101

Well contents: 33uL polymerase MM, 1uL each oligo, 0.5uL template DNA

  • Thermocycler program 4K55

Lane Part Polymerase Fwd oligo Rev oligo Predicted Size Acceptable?
1Self Lysis Device(Bjh2294)PhusionigemTen007G001012540Very Faint, but visible
2Self Lysis Device(Bjh2294)ExpandigemTen007G001012540No, nothing visible
3Self Lysis Device(Bjh2294)TaqigemTen007G001012540Very Faint, but visible
41601AC plasmidPhusionigemTen009G001013220 No, nothing visible
51601AC plasmidExpandigemTen009G001013220No, nothing visible
61601AC plasmidTaqigemTen009G001013220No, 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

Well contents: 33uL polymerase MM, 1uL each oligo, 0.5uL template DNA

Well Part Polymerase Fwd Oligo Rev Oligo
1B10sbb10ExpandigemTen014igemTen016
2iGEM10_009 AExpandigemTen015igemTen018
3iGEM10_008d ExpandigemTen017igemTen020
4iGEM10_017 AExpandca998igemTen022
5iGEM10_016aExpandigemTen021igemTen024
6pB6 (BAC)ExpandigemTen025igemTen026

Conor McClune 15:13, 14 June 2010 (EDT)

Gibson Assembly for iGEM10_007

Components

  • Parts (assembled in this order):
    • iGEM10_001
    • <piggybac!> (part name B10sbb04)
    • iGEM10_002
    • iGEM10_003
    • Self Lysis Device (part name Bjh2294)
  • Vector:
    • pBjh1601AC

Parts PCR

  • I have already PCR-magnified iGEM10_002 and iGEM10_003 with the appropriate Gibson oligos I designed

Procedure
Well contents:

  • 50μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA
Wells Part Fwd oligo Rev oligo
1iGEM10_001 aca998igemTen002
2iGEM10_001 bca998igemTen002
3iGEM10_001 cca998igemTen002
4<piggybac!igemTen001igemTen004
5Self Lysis Device(Bjh2294)igemTen007G00101
61601AC plasmid igemTen009igemTen010
  • Run on PCR program 4K55

Gel Results

Lane Part Fwd oligo Rev oligo Predicted Size Acceptable?
1iGEM10_001 aca998igemTen0021360 No, not visible
3iGEM10_001 bca998igemTen0021360No, not visible
5iGEM10_001 cca998igemTen0021360No, not visible
7<piggybac!igemTen001igemTen0041820No, not visible
9Self Lysis Device(Bjh2294)igemTen007G001012540No, not visible
111601AC plasmidigemTen009igemTen0103220No, not visible

2nd Attempt at PCR: Using Expand Polymerase

  • 33μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA
Wells Part Fwd oligo Rev oligo
1iGEM10_001 aca998igemTen002
2iGEM10_001 bca998igemTen002
3iGEM10_001 cca998igemTen002
4<piggybac!igemTen001igemTen004
5Self Lysis Device(Bjh2294)igemTen007G00101
61601AC plasmid igemTen009igemTen010
  • Ran on 5K55 and left on thermocycler overnight (see gel results on tomorrow's notebook entry)

Conor McClune 14:11, 11 June 2010 (EDT)

Oligo and Composite Parts Test PCR #2 (continued from yesterday)

15uL of PCR product run on gel:

Lane Part Fwd Oligo Rev Oligo Predicted Size Matches Gel Results?
1iGEM10_002digemTen003G00101500yes
2iGEM10_002dca998igemTen006470yes
3iGEM10_002dca998G00101570yes
4iGEM10_003bigemTen005G001011190yes
5iGEM10_003bca998igemTen0081160yes
6iGEM10_003bca998G001011260yes
7iGEM10_003digemTen005G001011190yes
8iGEM10_003dca998igemTen0081160yes
9iGEM10_003dca998G001011260yes



Oligo and Composite Parts Test PCR #3

Procedure Well contents:

  • 50μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA
well Polymerase Part Fwd Oligo Rev Oligo
1TaqiGEM10_002digemTen003igemTen006
2PhusioniGEM10_002digemTen003igemTen006
3TaqiGEM10_003bigemTen005igemTen008
4PhusioniGEM10_003bigemTen005igemTen008
  • Wells run on 2K55 on thermocycler.

Gel 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

Lane Polymerase Part Fwd Oligo Rev Oligo Predicted Size Matches Gel Results?
1TaqiGEM10_002digemTen003igemTen006400yes, strong band
2PhusioniGEM10_002digemTen003igemTen006400yes, weaker band
3TaqiGEM10_003bigemTen005igemTen0081100yes, strong band
4PhusioniGEM10_003bigemTen005igemTen0081100yes, weaker band
5n/apB640,000possible band at 40kb, but very low conc.

Gel Purification of iGEM10_002 and iGEM10_003

  • cut out Phusion transcribed bands:
    • band at 400 in lane 2
    • band at 1100 in lane 4
  • followed this protocol and placed the purified PCR products of iGEM10_002 and iGEM10_003 in the working box

PDD Part Preparation

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

Conor McClune 13:23, 10 June 2010 (EDT)

Negative Controls from Yesterday's Competent Cells

  • plates: Amp,Kan,Cam,Spec
  • positions: 1=pBca1256-Bjh2343, 2=pBca1256-Bjh2344, 3=pBca1256-Bjh1934, 4=MG1655



Plating yesterdays BAC tranformations failed

Retransformation

  • Transforming pB6 BAC into pBca1256-Bjh2343, pBca1256-Bjh2344 and pBca1256-Bjh1934
  • adjustments to 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



Oligo and Composite Parts Test PCR 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



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:

  1. iGEM10_002d+igemTen003+G00101
  2. iGEM10_002d+ca998+igemTen006
  3. iGEM10_002d+ca998+G00101
  4. iGEM10_003b+igemTen005+G00101
  5. iGEM10_003b+ca998+igemTen008
  6. iGEM10_003b+ca998+G00101
  7. iGEM10_003d+igemTen005+G00101
  8. iGEM10_003d+ca998+igemTen008
  9. iGEM10_003d+ca998+G00101

PCR run and products kept at 16*C in the thermocycler overnight

Conor McClune 15:08, 9 June 2010 (EDT)

Created -80 Stocks

  • used this protocol to make -80 stocks for
    • 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

  • for pBca1256-Bjh1934 in MG1655,Bjh2343 in MG1655 and Bjh2344 in MG1655:
    • added 500μL of overnight-grown culture to 50mL Spec LB in an Erlenmeyer flask
  • for MG1655:
    • 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)
  • both flasks put on shaker in lab at 12:00
  • 2:00pm
    • pBca1256-Bjh1934,Bjh2343 and Bjh2344 (all in MG1655) have all grown up well, but not MG1655 (probably because of the glycerol)
      • MG1655 was place back in the incubator
      • 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 the supernatant
        • added 2.5ml TSS solution to each of the three vials
        • made 98uL aliquots into 25 chilled eppendorf tubes for storage (25 tubes for each of pBca1256-Bjh1934,Bjh2343 and Bjh2344)
  • Transformed BAC into 1, 2, 3.
  • Plated on Cam, Spec plates.
  • 3:30
    • 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

Conor McClune 13:52, 8 June 2010 (EDT)

  • The five plates from yesterday (shown below) grew up well. We picked two colonies from the following:
    • 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
iGEM10_002 (part 11 next to 6 in AC vector): Expected band length: about 550bp
iGEM10_003 (part 20 next to 5 in CK vector): Expected band length: about 1250bp

Image:6-8-10.jpg
Colony PCR
Lane 1 iGEM10_003a (should be 1250bp)
Lane 2 iGEM10_003b (should be 1250bp)
Lane 3 iGEM10_003c (should be 1250bp)
4 iGEM10_003d (should be 1250bp)
5 iGEM10_001a (should be 1500bp)
6 iGEM10_001b (should be 1500bp)
7 iGEM10_001c (should be 1500bp)
8 iGEM10_001d (should be 1500bp)
9 iGEM10_002a (should be 550bp)
10 iGEM10_002b (should be 550bp)
11 iGEM10_002c (should be 550bp)
12 iGEM10_002d (should be 550bp)

We will choose the following samples to miniprep tomorrow:
iGEM10_001c,d
iGEM10_002c,d
iGEM10_003a,b

  • 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

Conor McClune 14:38, 7 June 2010 (EDT)


  • (with Tahoura)
  • 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
  • Plated pB6 (BAC), place in incubator. Needs to be picked and miniprepped tomorrow.
  • Transformed pBca1256-Bjh1934 into MG1655. Used procedure from (1)
  • Attempted to enter parts into clotho --> would not save

First step of Manual Assembly

Digestion

Lefty MM added to 9+8, 7+11, 9+20
Right MM added to 7+23, 8+6, 7+5
They should be done incubating at 3:48pm.


Lefty MasterMix (for 4uL miniprep DNA)

  • 4uL water
  • 1uL of NEB2
  • 0.5uL XhoI
  • 0.5uL BamH1

Righty MasterMix (for 4uL miniprep DNA)

  • 4uL water
  • 1uL of NEB2
  • 0.5uL XhoI
  • 0.5uL BglII
  • note: righty digestions had different volumes, which seemed suspicious, so the digestion was repeated with 1uL of each enzyme for 30 min.

Zymo

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.

Ligation:

Followed this protocol with the following amendments:
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
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