IGEM:MIT/2007/Notebook/2007-7-19

Agenda for Thursday, 7/19

• Make permanent glycerol stocks of 8 tubes
• AK/AL list
• Colony PCR on yesterday's plated transformation (F2620+B0034+CPX in pSB1AC3)

Colony Count of Yesterday's Plates

TK's DH5a transformed with F2620+B0034+CPX in pSB1AC3

Key:

• (+) contains DH5a transformed with digested and ligated parts
• (-) contains DH5a transformed with only digested and ligated pSB1AC3
• BH the plate is labeled with Bernice's initials
• JH the plate is labeled with Jess's initials

Plate                              #Colonies     Notes
(+) Cm        BH                   ~50           A couple satellite colonies
(+) Cm + Kan  BH                   1             This plate hardened imperfectly
(+) Cm        JH                   ~64
(+) Cm + Kan  JH                   3             On the lip of the plate
(-) Cm                             9             
(-) Cm + Kan                       0

Colony PCR of Yesterday's 3A Transformation

• Used 8 colonies picked from (+) Cm + Kan JK plate
• Streaked those 8 colonies also onto Cm and Cm + Kan plates for antibiotic screening
• Used the OWW Knight Protocol for Colony PCR (see below)

Colony PCR Protocol (OWW, Knight)

1. Prepare one sterile 0.6mL tube with 20 µl ddH20 for each colony
2. Prepare LB-agar plate with appropriate antibiotic to use as index plate
3. Pick single colony using a pipetman with sterile tip. The pipettor should be set to 3µl
4. Innoculate tip with colony into tube. Pipet up and down to ensure cells are transferred to tube. Pipet 3µl of cells suspended in water onto index plate.
5. Repeat for as many colonies as you intend to pick

Reaction Mixture:

• 9µl PCR supermix
• 0.25 µl 40µM VF2
• 0.25 µl 40µM VR
• 0.5 µl colony template

PCR conditions:

1. 95C for 15 mins
2. 94C for 30secs
3. 56C for 30secs
4. 68C for 1 min per kb of expected product
5. Repeat 2-4 39 times
6. 68C for 20 mins
7. 4C forever

• Run a gel to determine amplification product length
  • 5 mins @ 40V, remainder @ 85V
  • Used 50ml gel with two rows of 6-wells each
  • Row 1, well 1: 1kb ladder
  • Row 1, wells 2-5: Colony PCR samples #1-4
  • Row 2, well 1: 1kb ladder
  • Row 2, wells 2-5: Colony PCR samples #5-8

The resulting gel

Permanent Glycerol Stocks of DB3.1, F2620, B0034 and E1010

• SK: Made eight cryogenic vials of glycerol stock; all have 1 mL 40% glycerol + 1 mL liquid sample
• All vials in the white sticker labeled "iGEM 2007 DH5alpha" -80 degree freezer box
• DB3.1, 2 vials = original with pSB3K3 (K+)
• F2620, 2 vials = transformed (A+)
• B0034, 2 vials = transformed (A+)
• E1010, 2 vials = transformed (K+)

Liquid Culture of BBa_I13500

• Last night's growth was unsuccessful, so it was redone

Made More Plates

• just LB: 10 plates
• Cm+Amp: 30 plates
  • Cm: 50 µg/ml
  • Amp: 30 µg/ml

Protocol for de-phosphorylating DNA before ligating

• Add 1 ul Calf Intestinal Phosphatase or Alkaline Phosphatase
• 37 C for an hour
• Heat Kill
• Purify on Gel