IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/18

From OpenWetWare

Jump to: navigation, search
iGEM Project name 1 Main project page
Previous entry      Next entry

QS Track

Eric F.

Transformation of cells ligated by Phillip

  • Began thawing 6 aliquots of competent cells at 1635
  • 6 Aliquots were made up of
    • 2x α1
    • 3x α2
    • 1x B2
  • The aliquots were labeled to match the labeling from earlier in the experiment
    • "α1 1-1" corresponded to ligation 1
    • "α1 1-2" corresponded to ligation 2
    • "α2 2-3" corresponded to ligation 3
    • "α2 2-4" corresponded to ligation 4
    • "α2 2-5" corresponded to ligation 5
    • "B2 B-C" corresponded to the backbone psB1C3 which was used in the previous set of ligation experiments, this final aliquot had several purposes
      • If it worked, it would be a positive control, and prove the problem in the previous ligation was not the backbone
      • If it did not work and nothing else worked, it could indicate the transformation failed
      • If it did not work and other things did work, it could indicate why the first ligation failed
  • Began adding ligation mix at 1650
  • Finished adding ligation mix at 1656
  • Made a new tube of LB Broth only - old one looked cloudy
  • Note: α2 2-5 may have been contaminated by glove/tip/inside of tube incident
  • Began 1 hour incubation at 1734
  • Removed from incubator at 1839
  • Spread 450 μL on each plate (rather than 500), and let them dry in that air next to a bunsen burner for 10 minutes
  • Placed in Lagally lab Biohazard room 37 degree incubator
  • Note: Plate α2 2-4 appears to have a small piece of debris on it. It is circled with black marker
  • Ligation mix stored in 4 degree fridge
  • Chloro plates went in the incubator at 1900, must be removed by 1300 June 19, 2010

DspB Track

Marianne, Vicki

Resuspension of Primers

Primers arrived: Upstream primer: (with His6-tag)

5' - GTT TCT TCG CGG CCG CTT CTA GAT GCA TCA TCA TCA TCA TCA TAA AAA AGC AAT TA - 3'
Tm = 66.9 degrees C
Total: 56 bp; 16 bp to hybridize, 18 bp of His, rest is RE bp (EcoR1 is not included)

Upstream primer: (no His6-tag)

5' - GTT TCT TCG CGG CCG CTT CTA GAT GAA AAA AGC AAT TAC - 3'
Tm = 64.2 degrees C
Total: 39 bp; 17 to hybridize, rest is RE bp (EcoR1 is not included)

Downstream primer:

5' - GTT TCT TCC GGC CGC TAC TAG TAC TAA TGC GAT TTC GGA - 3'
Tm = 65.9 degrees C
Total: 39 bp; 16 bp to hybridize, rest is RE bp (PstI is not included)

Resuspended primers in TE buffer (found in Invitrogen Kit) to a concentration of 100uM

Primers:

  1. upstream + His dspB: 1.616mL of TE buffer + 161.6nm of primer
  2. downstream dspB: 1.664mL of TE buffer + 166.4nm of primer
  3. upstream dspB: 1.689mL of TE buffer + 168.9nm of primer

Stored in -20degree C fridge


Personal tools