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



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