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
1 iGEM10_013 #14 Eco/Bam 6204
2 iGEM10_013 #16 Eco/Bam 6204
3 iGEM10_007 #11 Eco/Bam 7060
4 iGEM10_007 #13 Eco/Bam 7060
5 Bth030 Eco/Bam 5850

Analytical PCR

Well Sample Part Checked fwd oligo rev oligo expected size
1 iGEM10_013 #14 iGEM10_001 ca998 igemTen002 1435
2 iGEM10_013 #14 B10sbb10 igemTen034 igemTen036 1026
3 iGEM10_013 #14 iGEM10_009 igemTen035 igemTen018 279
4 iGEM10_013 #14 iGEM10_008 igemTen017 igemTen020 1057
5 iGEM10_013 #14 Bjh2294 w/ fwd oligo 19 igemTen019 G00101 2607
6 iGEM10_013 #16 iGEM10_001 ca998 igemTen002 1435
7 iGEM10_013 #16 B10sbb10 igemTen034 igemTen036 1026
8 iGEM10_013 #16 iGEM10_009 igemTen035 igemTen018 279
9 iGEM10_013 #16 iGEM10_008 igemTen017 igemTen020 1057
10 iGEM10_013 #16 Bjh2294 w/ fwd oligo 19 igemTen019 G00101 2607

ConorGel33.jpg

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

ConorGel34.jpg
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
1 iGEM10_007 #11 iGEM10_001 ca998 igemTen002 1435
2 iGEM10_007 #11 (B10sbb04) igemTen001 igemTen004 1788
3 iGEM10_007 #11 iGEM10_002 igemTen003 igemTen006 366
4 iGEM10_007 #11 iGEM10_003 igemTen005 igemTen008 1064
5 iGEM10_007 #11 Self Lysis Device (Bjh2294) igemTen007 G00101 2607
6 iGEM10_007 #13 iGEM10_001 ca998 igemTen002 1435
7 iGEM10_007 #13 (B10sbb04) igemTen001 igemTen004 1788
8 iGEM10_007 #13 iGEM10_002 igemTen003 igemTen006 366
9 iGEM10_007 #13 iGEM10_003 igemTen005 igemTen008 1064
10 iGEM10_007 #13 Self Lysis Device (Bjh2294) igemTen007 G00101 2607

ConorGel30.jpg

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
1 iGEM10_013 #14 iGEM10_001 ca998 igemTen002 1435
2 iGEM10_013 #14 B10sbb10 igemTen034 igemTen036 1026
3 iGEM10_013 #14 iGEM10_009 igemTen035 igemTen018 279
4 iGEM10_013 #14 iGEM10_008 igemTen017 igemTen020 1057
5 iGEM10_013 #16 iGEM10_001 ca998 igemTen002 1435
6 iGEM10_013 #16 B10sbb10 igemTen034 igemTen036 1026
7 iGEM10_013 #16 iGEM10_009 igemTen035 igemTen018 279
8 iGEM10_013 #16 iGEM10_008 igemTen017 igemTen020 1057
9 iGEM10_013 #14 Bjh2294 w/ fwd oligo 19 igemTen019 G00101 2607
10 iGEM10_013 #16 Bjh2294 w/ fwd oligo 19 igemTen019 G00101 2607
  • 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.

ConorGel32.jpg

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)
ConorGel28.jpg ConorGel29.jpg
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

ConorGel25.jpg

  • igem10_020 #12

ConorGel26.jpg
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)

ConorGel27.jpg

  • 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_001 1335 bright 0.5 0.714285714
B10sbb10 1026 bright 0.5 0.714285714
iGEM10_009 279 dim 0.5 0.714285714
iGEM10_008 1057 bright 0.5 0.714285714
Bjh2294 w/ fwd oligo 19 2507 bright 0.5 0.714285714
p1601AC (vector) 3183 medium bright 1 1.428571429
total 3.5 5
  • 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
1 4uL iGem10_020 Gibson Colony #12 1uL NEB3 1uL Eco 1uL Bgl 4ul
2 4uL iGem10_020 Gibson Colony #15 1uL NEB3 1uL Eco 1uL Bgl 4ul
3 8uL iGem10_001 c 1uL NEB2 2uL Eco 2uL Bam 8ul


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
1 10uL iGem10_020 Gibson Colony #12 10uL iGem10_001 c 10uL Ligase Buffer 5uL T4 Ligase 65ul
2 10uL iGem10_020 Gibson Colony #15 10uL iGem10_001 c 10uL Ligase Buffer 5uL T4 Ligase 65ul

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
1 iGEM10_007 #3 ca56 igemTen002 256
2 iGEM10_007 #3 ca56 igemTen004 2044
3 iGEM10_007 #3 igemTen003 igemTen008 1462
4 iGEM10_007 #3 igemTen007 G00101 2635
5 iGEM10_007 #7 ca56 igemTen002 256
6 iGEM10_007 #7 ca56 igemTen004 2044
7 iGEM10_007 #7 igemTen003 igemTen008 1462
8 iGEM10_007 #7 igemTen007 G00101 2635

Gel Results
ConorGel23.jpg

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 B10sbb10 igemTen034 igemTen036 Expand 1050
2 iGEM10_009 igemTen035 igemTen018 Expand 300
3 B10sbb10 igemTen034 igemTen036 Phusion 1050
4 iGEM10_009 igemTen035 igemTen018 Phusion 300

'Gel Results'
ConorGel24.jpg

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:

ConorGel22.jpg 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):

ConorGel19.jpg

    • plate 2(colonies 9-16):

ConorGel20.jpg

  • 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) :

ConorGel21.jpg

  • 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:

ConorGel18.jpg

  • 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:

ConorGel17.jpg

  • 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_017 yes 1546 bright 0.5 0.833333333
iGEM10_016 yes 858 medium dim 1 1.666666667
Bjh2294 with fwd oligo 23 yes 2507 bright 0.5 0.833333333
p1601AC (vector) yes 3183 medium bright 1 1.666666667
total: 3 5
  • 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

ConorGel16.jpg

  • 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_001 yes 1335 bright 0.25 0.3125
(B10sbb04) yes 1788 bright 0.25 0.3125
iGEM10_002 yes 366 dim 0.5 0.625
iGEM10_003 yes 1064 dim 1 1.25
Self Lysis Device (Bjh2294) yes 2507 medium bright 1 1.25
pBjh1601AC (vector) yes 3183 medium bright 1 1.25
total 4 5
  • 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

ConorGel14.jpg

  • 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
1 Bjh2294 Phusion igemTen19 G00101 4K55
2 Bjh2294 Phusion igemTen23 G00101 4K55
3 Bjh2294 Phusion igemTen19 G00101 4K45
4 Bjh2294 Phusion igemTen23 G00101 4K45

Gel Results (the lane numbers correspond the the well numbers above)
ConorGel15.jpg

  • 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_001 0.833
<piggybac!(B10sbb04) 0.833
iGEM10_002 0.833
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)

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

ConorGel11.jpg
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

ConorGel13.jpg

  • 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
1 sbb10 Phusion igemTen14 igemTen16
2 sbb11 Expand igemTen14 igemTen16
3 sbb12 Phusion igemTen14 igemTen16
4 sbb13 Expand igemTen14 igemTen16

Wells 1,2 run on program COL2K
Wells 3,4 run on program 2K45
ConorGel12.jpg

  • 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
ConorGel9.jpg

Well Part Polymerase Fwd Oligo Rev Oligo Size Expected Acceptable?
1 B10sbb10 Expand igemTen014 igemTen016 260 Very faint
2 iGEM10_009 A Expand igemTen015 igemTen018 310 Yes
3 iGEM10_008d Expand igemTen017 igemTen020 1090 Yes
4 iGEM10_017 A Expand ca998 igemTen022 1650 Yes
5 iGEM10_016a Expand igemTen021 igemTen024 890 Yes
6 pB6 (BAC) Expand igemTen025 igemTen026 1000 Yes
  • 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
1 B10sbb10 Phusion igemTen014 igemTen016
2 B10sbb10 Expand igemTen014 igemTen016
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)
Well Part Polymerase Fwd Oligo Rev Oligo Size Expected
1 B10sbb10 Phusion igemTen014 igemTen016 260
2 pBjh1601 Phusion igemTen009 ss13r 3183
3 pBjh1601 Expand igemTen009 ss13r 3183

Gel Results
ConorGel10.jpg

Lane Part Polymerase Fwd Oligo Rev Oligo Size Expected Acceptable?
1 B10sbb10 Phusion igemTen014 igemTen016 260 no
2 B10sbb10 Expand igemTen014 igemTen016 260 no
3 Bjh2294 Phusion igemTen007 G00101 2540 Probably just template DNA
4 Bjh2294 Expand igemTen007 G00101 2540 no
5 Bjh2294 (PCR Product) Phusion igemTen007 G00101 2540 yes
6 - - - - -
7 B10sbb10 Phusion igemTen014 igemTen016 260 no
8 pBjh1601 Phusion igemTen009 ss13r 3183 yes
9 pBjh1601 Expand igemTen009 ss13r 3183 no
  • 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.

ConorGel6.jpg

  • gel looked messy, so I ran it again:

ConorGel7.jpg

Lane Part Fwd oligo Rev oligo Predicted Size 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

Well Part Polymerase Fwd oligo 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

ConorGel8.jpg

Lane Part Polymerase Fwd oligo Rev oligo Predicted Size 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

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

Well Part Polymerase Fwd Oligo Rev Oligo
1 B10sbb10 Expand igemTen014 igemTen016
2 iGEM10_009 A Expand igemTen015 igemTen018
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

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
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
  • Run on PCR program 4K55

Gel Results
ConorGel5.jpg

Lane Part Fwd oligo Rev oligo Predicted Size 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
Wells Part Fwd oligo 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)

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:
ConorGel2.jpg

Lane Part Fwd Oligo Rev Oligo Predicted Size 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



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
1 Taq iGEM10_002d igemTen003 igemTen006
2 Phusion iGEM10_002d igemTen003 igemTen006
3 Taq iGEM10_003b igemTen005 igemTen008
4 Phusion iGEM10_003b igemTen005 igemTen008
  • Wells run on 2K55 on thermocycler.

Gel Results
ConorGel3.jpg

  • possible DNA spilling from lane 4 to lanes 3&5, thus I repeated the gel run to avoid a false positive in lane 5

ConorGel4.jpg

Lane Polymerase Part Fwd Oligo Rev Oligo Predicted Size 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
4 Phusion iGEM10_003b igemTen005 igemTen008 1100 yes, weaker band
5 n/a pB6 40,000 possible 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

NegControlAmp.jpg NegControlKan.jpg NegControlCam.jpg NegControlSpec.jpg

Plating yesterdays BAC tranformations failed
BAC2343.jpg BAC2344&1934.jpg
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

ConorGel1.jpg

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

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)

Jin Notes1.JPG

  • (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