Sam:LabNotes/Microbiome-new/2009-2-25: Difference between revisions

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='''High-throughput single gene PCR validation for extracted E.coli and MDA amplicons'''=


==Procedure==
*Primer dilution x 8 (primer E-1~E-10)
                      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
Templates            Dilution        Description   
-----------------------------------------------------------------
Row A: E.coli gDNA-1  - 1/50  - Extracted using Promega Wizard kit
Row B: E.coli gDNA-2  - 1/50  - Extracted using Promega Wizard kit
Row C: A1 MDA amplicon - 1/50  - Whole single E.coli MDA on 3-18-09
Row D: A3 MDA amplicon - 1/50  - Whole single E.coli MDA on 3-18-09
Row E: A4 MDA amplicon - 1/50  - Whole single E.coli MDA on 3-18-09
Row F: A5 MDA amplicon - 1/50  - Whole single E.coli MDA on 3-18-09
 
Each template was tested on eight primers (E-1 ~ E-8)
*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==
  [[Image:ZhangLab_2 2009-03-26 09hr 54min-annotate2.jpg|700px]]
  [[Image:ZhangLab_2 2009-03-26 10hr 37min-annotate.jpg|700px]]
 
  Primer(amplicon size)
==Discussion==
*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.

Latest revision as of 21:26, 26 March 2009