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.