Sam:LabNotes/Human/2009-3-24: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
(New page: =High through-put single gene PCR validation= ==Objective== *Since MPCR is easily biased by different template dilution and primer competition, the single gene validation is more reliabl...)
 
>Sam Chiang
Line 27: Line 27:


*Exp design
*Exp design
Row A: p-pos1 (D7) 1/15 dilugion
Row A: p-pos1 (D7) 1/15 dilugion
Row B: p-pos2 (D8) original
Row B: p-pos2 (D8) original
Row C: p-pos2 (D8) 1/25 dilution
Row C: p-pos2 (D8) 1/25 dilution
Row D: gDNA        3 ng/uL
Row D: gDNA        3 ng/uL

Revision as of 07:28, 25 March 2009

High through-put single gene PCR validation

Objective

  • 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

  • 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