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