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

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=High through-put single gene PCR validation=
* '''[[Sam:LabNotes/Human/2009-3-24 exp1|High through-put single gene PCR validation]]'''


 
* '''[[Sam:LabNotes/Human/2009-3-12 exp2|High through-put single gene PCR validation with template dilution on 1/25, 1/50, 1/100 ]]'''
==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
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==
  [[Image:ZhangLab_2 2009-03-24 13hr 28min-annotate.jpg|600px]]
  [[Image:ZhangLab_2 2009-03-24 14hr 17min-annotate.jpg|600px]]
 
==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

Revision as of 06:46, 26 March 2009