Sam:LabNotes/Microbiome-new/2009-3-25
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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) E-1 TopA(260), E-2 ExuR(232), E-3 GlyS(245), E-4 OmpA(135), E-5 CadA(258) E-6 PcnB(205), E-7 HolA(121), E-8 Exo(195), E-9 Nth(289), E-10 EutC(233)- * E-9 and E-10 are not used in the current exp Amplicon sizes were estimated using UCSC e-PCR against E.coli K12 strain genome
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. The high representation rate might be due to the small size of E.coli genome compared to mammalian cells, or the underestimated the bacteria number in the bacteria dilution.