Sam:LabNotes/Human/2009-3-24 exp1

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High through-put single gene PCR validation[edit]

Objective[edit]

  • Since MPCR is easily biased by different template dilution and primer competition, the single gene validation is more reliable and practical.
  • Design a high through-put method:
    • Test all 23 genes at the same time(Using 26-well comb gel electrophoresis)
    • Using multi-channel pipette for fast primer/enzyme loading
    • Using diluted template 1/25 (for 23 genes - whole genome set primers)

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
  • Dilute template into 1/25 (Add 1 uL template into 24 uL H2O)
 Template 1 (p-pos1): 03-12-09  D7 (10 cell)–GM18506 from test of L4N6 human MDA – single cell
 Template 2 (p-pos2): 03-12-09  D8 ( 5 cell)–GM18506 from test of L4N6 human MDA – single cell
 Template 3   (gDNA): 3 ng/uL
  • Exp design
Row A: p-pos1 (D7) 1/15 dilugion
Row B: p-pos2 (D8) original
Row C: p-pos2 (D8) 1/25 dilution
Row D: gDNA        3 ng/uL

Each template was tested on eight primers (1-1 ~ 8-1)
  • Program: T52C25 (Tm=52C, for 25 cycles of ampplification)
  • Gel electrophoresis
    • 2% agarose gel, midium tray with 26-well comb
    • Sample (7uL) + Loading buffer (1.5 uL)
    • Run at 135V for 25 min

Results[edit]

 File:ZhangLab 2 2009-03-24 13hr 28min-annotate.jpg 
 File:ZhangLab 2 2009-03-24 14hr 17min-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 positive control shows the weak bands in expected size. The amplification cycle may need to be increased in the future experiment.
  • Suprisingly, none of p-pos templaes showed bands. The results of D7 is similar to the results from previous test on 3-22-09