Sam:LabNotes/Microbione/2009-3-3

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Human genome screening primer set - quality validation[edit]

Objective[edit]

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

Samples & Materials[edit]

  • 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[edit]

  • 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 medium 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
  • Repeat the same design to perform PCR on primer set2 (23 reactions)


Results[edit]

  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 (1-1 -> 8-1, ladder, 9-1 -> 16-1, ladder, 17-1 -> 22-1, X-1, Y-1)
  • Lower: Test by primer set2 (1-2 -> 8-2, ladder, 9-2 -> 16-2, ladder, 17-2 -> 22-2, X-2)
  • Primer name (amplicon size)
1-1   (270)	4-1   (383)	7-1   (242)	10-2   (299)	13-2   (1102)	16-2   (1324)	19-1   (1007)	22-2  (1166)
1-2   (676)	4-2   (896)	7-2   (498)	10-1   (214)	13-1   (679)	16-1   (806)	19-2   (1512)	22-1  (757)
2-1   (540)	5-1   (413)	8-2   (231)	11-1   (131)	14-1   (598)	17-2   (1240)	20-1   (174)	x-2   (839)
2-2   (649)	5-2   (530)	8-1   (230)	11-2   (525)	14-2   (756)	17-1   (419)	20-2   (584)	x-1   (147)
3-1   (235)	6-2   (1050)	9-1   (373)	12-1   (984)	15-1   (297)	18-2   (288)	21-1   (200)	y-1   (122)
3-2   (1323)	6-1   (299)	9-2   (907)	12-2   (1689)	15-2   (1422)	18-1   (171)	21-2   (346)	


Discussion & Suggestion[edit]

  • Except primer set1 chromosome X and Y which have only weak bands, all primers generate strong band with good amplification at 52C
  • The PCR cycle is too many -> saturation of amplicons
  • The primer concentration used in PCR is too high -> serious primer dimer seen on the gel