Sam:LabNotes/Microbione/2009-3-2

From ZhangLabWiki
Revision as of 07:47, 5 March 2009 by >Sam Chiang (→‎Results)
Jump to navigation Jump to search

Human genome screening primer set - quality validation

Objective

  • Human whole genome primer sets were obtained from Professor (2 sets).
  • The primer set were used to confirm the existance of choromosomes in MDA amplicon using multiplex PCR.
  • Individual validation of each primer is required before performing multiplex PCR.


Samples & Materials

  • Enzyme - Taq 2X master mix (NEB)
  • Templates (1 uL): 1 ng/uL human gDNA (NEB)
  • Primers: All primers are diluted to 100 uM, and mixed basd on the gene (Forward 20 uL + Reverse 20 uL + H2O 160 uL)
    • Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns)
    • Set2: 1-2 ~ 22-2 and X-2 (23 rxns)


Procedures

  • Thaw the Taq2X enzyme and primers.
  • Dilute all MDA template. Prepare diluted Pos template (1 ng/uL)
  • Set up PCR program (GENE59, GENE52).
  • Master Mix - x 2 (Primers are various)
    • Total reactions: 12 rxns
           1 rxn        24+3 rxn
     ---------------------------
     H2O      3.5       94.5
     Template 1.0       27.0  
     Taq 2X   5.0      135.0
     ---------------------------
              9.5      256.5 (uL)  256.5/27=9.5 


  • Transfer 9.5 uL of master mix into each of 24 PCR tubes (8-well strip)
  • Transfer the 0.5 uL primer into each reaction
  • Perform PCR reaction in thermocycler: program GENE52 (Tm52, 30 cycles)
  • Gel Electrophoresis
    • 1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> 2 midium tray with 26-well comb
    • Run at 135 V for 18 min.
    • Loading: Sample 7 uL + 2 uL loading buffer -> 7 uL. Diluted ladder 7 uL
    • Loading order (Left to right): 1-1 -> 22-1 -> X-1 ->Y-1 -> Low mass ladder
  • Repeat the same design to perform PCR on primer set2 (23 reactions)

Results

  File:ZhangLab 2 2009-03-03 15hr 01min-set1-new.jpg 
  File:ZhangLab 2 2009-03-03 15hr 01min-set2.jpg
  
  • Upper: Test by primer set1 (chromosome 1 ->8, ladder, 9->16, ladder, 17-22, X, Y)
  • Lower: Test by primer set2 (chromosome 1 ->8, ladder, 9->16, ladder, 17-22, X)

Discussion