Sam:LabNotes/Microbiome-new/2009-2-25

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Revision as of 18:12, 26 March 2009 by >Sam Chiang (→‎Discussion)
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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

 File:ZhangLab 2 2009-03-26 09hr 54min-annotate2.jpg 
 File:ZhangLab 2 2009-03-26 10hr 37min-annotate.jpg
 
 Primer(amplicon size)

Discussion

  • We accidently using PCR program with Tm=52 without checking the primers' desired Tm from previous study. However the amplication were still successful and no obvious un-specific amplification was found. It is possible that the lower Tm might be more suitable for amplyfying on MDA templates.
  • Both E.coli gDNA and MDA template (from whole single E.coli) came out pretty high representation rate for the eight genes (primers) we tested.