Sam:LabNotes/Human/2009-3-24 exp2

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High through-put single gene PCR validation with template dilution on 1/25, 1/50, 1/100[edit]

Procedure[edit]

  • Primer dilution x 8 (primer 1-1~1-8 from set-1)
                      1 rxn       20 rxn
    Primer (f+r)        0.5        10 uL
    H2O                 3.5        70 uL
    ------------------------------------
                        4.0        80 uL


  • Aliquots Taq2X enzymes into PCR tubes (100 uL) x 8 PCR tubes
  • Exp design
Row A: B7 - 1/25(from exp of test2 2-25-2009), amplicon from whole single cell 
Row B: B7 - 1/50 
Row C: B7 - 1/100
Row D: D5 - 1/25(from exp of test 2-27-2009), amplicon from 20 cells.
Row E: D5 - 1/50
Row F: D5 - 1/100
 
Each template was tested on eight primers (1-1 ~ 8-1)
  • Amplication: Program: T52C30 (Tm=52C, for 30 cycles)
  • Gel electrophoresis
    • 2% agarose gel, midium tray with 26-well comb
    • Sample (9uL) + Loading buffer (2 uL)
    • Run at 135V for 20 min

Results[edit]

 File:ZhangLab 2 2009-03-24 18hr 58min-annotate.jpg 
 File:ZhangLab 2 2009-03-25 10hr 51min-annotate.jpg
 
 Primer(amplicon size)
 
 1-1(270), 2-1(540), 3-1(235), 4-1(383), 5-1(413), 6-1(299), 7-1(242), 8-1(230)

Discussion[edit]

  • The dilution of template (from original MDA amplicon ~2000 ng/uL) into 1/25, 1/50 or 1/100 didn't affact the appearance of bands in the current experiment.
  • Amplication of 30 cycles improved the band intensity obviously.
  • Gel electrophoresis ran at 20 min is optimal for this screening.