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

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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

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


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