Berk2010-Conor

To Do:

• pick colonies of BAC/place in lb (Chlor)
• pick stage 1 assembly
• go through automated assembly to decide which strains we need to transform basic parts into (lefty or rightly)
• grow up pB6 cells for -80*C (add glycerol)

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

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