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
Procedure
- 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, 1/50, 1/100 dilution
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: 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
File:ZhangLab 2 2009-03-24 13hr 28min-annotate.jpg File:ZhangLab 2 2009-03-24 14hr 17min-annotate.jpg
Discussion
- 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