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

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dspB Track

PCR with His off the minipreps

Protocol: SOP PCR cycle program: dspB cycle

  • Changes: Initial step at 30s
PCR Master mix
Reagent1x rxn volume (uL)
5x rxn buffer5
10mM dNTP0.5
sdH2O9.15
Phusion polymerase0.1
MgCl22
DMSO - 5%1.25
gDNA3
Total25
  • Add 2uL of fw His + rev primers

Tubes: His,W

Gel verification

  • Protocol: biobrick "digest" protocol

Gel orientation:

Gel orientation
dspB His100bp ladderW

Machine conditions: 0.5x TBE buffer, 100V, 30min Results:

Restriction Digest

Restriction Digest
REAGENTS1 RXN VOLUME (uL)
Buffer 22
BSA0.2
DNAx
ddH20y
Total20
  • 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

PCR Master mix
Reagent1x rxn volume (uL)
5x rxn buffer5
10mM dNTP1
sdH2O6.65
Phusion polymerase0.1
MgCl22
DMSO - 5%1.25
gDNA3
Total25
  • 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

  • Protocol: biobrick "digest" protocol

Gel orientation:

Gel orientation
2_12_72_12W100bp ladderXE/SX/P

Machine conditions: 0.5x TBE buffer, 100V, 60min Results:

Restriction Digest

Restriction Digest
REAGENTS1 RXN VOLUME (uL)
Buffer 22
BSA0.2
DNA10
ddH205.8
Total20
  • 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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