Sam:LabNotes/Microbiome-new/2009-3-25: Difference between revisions
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>Sam Chiang |
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Templates Dilution Description | Templates Dilution Description | ||
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Row A: E.coli gDNA-1 - 1/50 - Extracted using Promega Wizard kit | Row A: E.coli gDNA-1 - 1/50 - Extracted using Promega Wizard kit (650 ng/uL) | ||
Row B: E.coli gDNA-2 - 1/50 - Extracted using Promega Wizard kit | Row B: E.coli gDNA-2 - 1/50 - Extracted using Promega Wizard kit (406 ng/uL) | ||
Row C: A1 MDA amplicon - 1/50 - Whole single E.coli MDA on 3-18-09 | 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 D: A3 MDA amplicon - 1/50 - Whole single E.coli MDA on 3-18-09 | ||
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Row F: A5 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) | Each diluted template(1 uL) was tested on eight primers (E-1 ~ E-8) | ||
*Amplication: Program: T52C30 (Tm=52C, for 30 cycles) | *Amplication: Program: T52C30 (Tm=52C, for 30 cycles) |
Revision as of 01:32, 27 March 2009
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 (650 ng/uL) Row B: E.coli gDNA-2 - 1/50 - Extracted using Promega Wizard kit (406 ng/uL) 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 diluted template(1 uL) 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.
- Optimal condition for Bac MDA amplicon validation: Tm=52C and 35 cycles of amplification.
- 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 MDA amplification.