IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/16

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PCR with His off the minipreps
Protocol: SOP PCR cycle program: dspB cycle
 * Changes: Initial step at 30s


 * Add 2uL of fw His + rev primers

Tubes: His,W

Gel verification
Gel orientation: Machine conditions: 0.5x TBE buffer, 100V, 30min Results:
 * Protocol: biobrick "digest" protocol

Restriction Digest

 * x = amount of DNA
 * DNA: NCC (no his on ccdB chlorbb)
 * For enzymes E/S, use 2uL of DNA
 * For enzymes X/P, use 2uL of DNA
 * For enzymes X, use 2uL of DNA
 * y = amount of ddH2O
 * Use appropriate amount of water given the amount of x DNA added

Digest for 2 hours

PCR Purification (Qiagen Kit)

 * PCR sample volume = 20uL
 * Need 100uL of Buffer PB
 * Nanodrop: 15.5ng/uL

PCR (Rafael's method) Rxn2 Attempt #3 @ 3 different annealing Temperature
Protocol: SOP


 * Add 3uL of fw His + rev primers

Tubes:
 * Column 1: 2_1
 * Column 7: 2_7
 * Column 12: 2_12


 * Use 'test' program by Rafael
 * Start: 7:19pm
 * End: 8:34pm

Gel verification
Gel orientation: Machine conditions: 0.5x TBE buffer, 100V, 60min Results:
 * Protocol: biobrick "digest" protocol

Restriction Digest

 * Digest His-dspB at X and P
 * Enzymes: add 1uL of XbalI and 1uL of PstI

Digest for 2 hours

Weight of X/P: 0.1548g


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