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

iGEM Project name 1

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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: upstream + His dspB: 1.616mL of TE buffer + 161.6nm of primer downstream dspB: 1.664mL of TE buffer + 166.4nm of primer upstream dspB: 1.689mL of TE buffer + 168.9nm of primer Stored in -20degree C fridge