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