Sam:LabNotes/Microbione/2009-3-2

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Revision as of 07:01, 5 March 2009 by >Sam Chiang (New page: =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 exi...)
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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)


Exp. Design

                                            Templates (A->H)
         ----------------------------------------------------------- 
                A2  A4  A5  A7  B1  B2  B4  B6  B7  C7  p-Pos  p-Neg
         -----------------------------------------------------------
  Primer P1 18S
         P2 2-1 
  Templates were tested in original and 1/10 dilution
        

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        12+2 rxn
     ---------------------------
     H2O     3.5       49.0 
     Primer  0.5        7.0  
     Taq 2X  5.0       70.0
     ---------------------------
             9.0      126.0 (uL)  126/14=9  


  • Transfer the 1 uL template to PCR tubes (8-well strip)
  • Transfer 9 uL of master mix into each reaction
  • Perform PCR reaction in thermocycler
    • Strip P1 goes to program GENE59 (Tm59, 30 cycles)
    • Strip P2 goes to 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 20 min.
    • Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL
    • Loading order (Left to right): A2->P-Neg (original dilution) -> laddr -> A2->P-Neg (1/10 dilution)


Results

  File:ZhangLab 2 2009-02-28 17hr 24min-Test3-Primer18S-annotate-new.bmp 
  File:ZhangLab 2 2009-02-28 18hr 12min-Test3-Primer2-1 annotate.bmp
  
  • Upper: Test by primer 18S (306 bp)
  • Lower: Test by primer 2-1 (540 bp)


Discussion

  • Even 1/10 dilutio didn't help improve the regular PCR performance a lot. The p-Pos results still looks very weak in prier 2-1 test
  • Pick up amplicons from B1 and B4 for 2nd MDA amplification