Berk2010-Conor

To Do:

• Finish creating competent cells:
  • 2343
  • 2344
  • 1256-1934
  • MG1655
• Miniprep cells for assembly of iGEM10_007
  • do Gibson PCR when plasmids arrive at 3pm
    • use 1601(Jin's assembly vector) and the strain 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)

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