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

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Revision as of 06:59, 26 March 2009 by >Sam Chiang (New page: ='''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 r...)
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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