Sam:LabNotes/Microbiome-new/2009-3-25: Difference between revisions

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


==Background==
*Ten pair of E.coli genes primers are ordered from IDT for E.coli MDA amplicon validation
**The reference of primers sequence: [[Media:2005_Genomic DNA amplification from a single bacterium - good.pdf|2005_Genomic DNA amplification from a single bacterium]]
Primer sequences
[[File:sam032510- ten Ecoli genes.jpg|700px]]


==Procedure==
==Procedure==
*Primer dilution x 8 (primer E-1~E-10)  
*Primer dilution x 8 (primer E-1~E-8)  
                       1 rxn      20 rxn
                       1 rxn      20 rxn
     Primer (f+r)        0.5        10 uL
     Primer (f+r)        0.5        10 uL
     H2O                3.5        70 uL
     H2O                3.5        70 uL
    Template            1.0        -     
    2X Taq enzyme (NEB) 5.0      100 uL     
     ------------------------------------
     ------------------------------------
                        4.0       80 uL
                        10.0       180 uL/20= 9uL -- template 1uL




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  Templates            Dilution        Description     
  Templates            Dilution        Description     
  -----------------------------------------------------------------
  -----------------------------------------------------------------
  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)
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   [[Image:ZhangLab_2 2009-03-26 10hr 37min-annotate.jpg|700px]]
   [[Image:ZhangLab_2 2009-03-26 10hr 37min-annotate.jpg|700px]]
    
    
   Primer(amplicon size)
   Gene name(amplicon size, bp)
   E-1 TopA(260), E-2 ExuR(232), E-3 GlyS(245), E-4 OmpA(135), E-5 CadA(258)
   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
   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==
==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.
*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 PCR amplification, run at 10 uL reaction, using regular Taq enzyme.
*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.
**94C 2m -> (94C 40s -> 52C 40s -> 72C 1m) x 35 -> 72C 10m -> 15C forever
*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 a MDA reaction.

Latest revision as of 21:03, 19 August 2011

Ten E.coli genes validation for extracted E.coli and MDA amplicons[edit]

Background[edit]

Primer sequences

File:Sam032510- ten Ecoli genes.jpg

Procedure[edit]

  • Primer dilution x 8 (primer E-1~E-8)
                      1 rxn       20 rxn
    Primer (f+r)        0.5        10 uL
    H2O                 3.5        70 uL
    Template            1.0         -      
    2X Taq enzyme (NEB) 5.0       100 uL       
    ------------------------------------
                       10.0       180 uL/20= 9uL -- template 1uL


  • 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[edit]

 File:ZhangLab 2 2009-03-26 09hr 54min-annotate2.jpg 
 File:ZhangLab 2 2009-03-26 10hr 37min-annotate.jpg
 
 Gene name(amplicon size, bp)
 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[edit]

  • 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 PCR amplification, run at 10 uL reaction, using regular Taq enzyme.
    • 94C 2m -> (94C 40s -> 52C 40s -> 72C 1m) x 35 -> 72C 10m -> 15C forever
  • 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 a MDA reaction.