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

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>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...)
 
>Sam Chiang
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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==
  [[Image:ZhangLab_2 2009-02-28 17hr 24min-Test3-Primer18S-annotate-new.bmp|800px]]
  [[Image:ZhangLab_2 2009-02-28 18hr 12min-Test3-Primer2-1 annotate.bmp|800px]]
 
*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

Latest revision as of 20:43, 8 March 2009