Berk2010-Conor: Difference between revisions

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*Run on PCR program 4K55 <br>
*Run on PCR program 4K55 <br>
'''Gel Results'''
'''Gel Results''' <br>[[Image: ConorGel4.jpg||400px]]
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{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Lane'''

Revision as of 15:59, 14 June 2010

To Do:

  • do Gibson PCR
    • for assembly of iGEM10_007, iGEM10_013 and iGEM10_020
    • use 1601(Jin's assembly vector) and the strain mc1061

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 igemTen007 G00101
6 1601AC plasmid igemTen009 igemTen010
  • Run on PCR program 4K55

Gel Results

Lane Part Fwd oligo Rev oligo Predicted Size Acceptable Size?
1 iGEM10_001 a ca998 igemTen002 1360
3 iGEM10_001 b ca998 igemTen002 1360
5 iGEM10_001 c ca998 igemTen002 1360
7 <piggybac! igemTen001 igemTen004 1820
9 Self Lysis Device igemTen007 G00101 2540
11 1601AC plasmid igemTen009 igemTen010 3220

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?
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

  • 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?
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



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 



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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