Sam:LabNotes/Microbione/2009-3-2: Difference between revisions

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=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==
  [[Image:ZhangLab_2 2009-03-03 15hr 01min-set1-new.jpg|600px]]
  [[Image:ZhangLab_2 2009-03-03 15hr 01min-set2.jpg|600px]]
 
*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==

Latest revision as of 20:43, 8 March 2009