Sam:LabNotes/Microbiome-new/2010-4-16: Difference between revisions

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*There are unspecific amplification. Unspecific amplication were difficul to be seen and removed by E-gel and still could be amplified in the 2nd round PCR.  
*There are unspecific amplification. Unspecific amplication were difficul to be seen and removed by E-gel and still could be amplified in the 2nd round PCR.  
**Sol.1 => Use the old school trick to do gel cutting on target band for sequencing.
**Sol.1 => Use the old school trick to do gel cutting on target band for sequencing.
**Sol. 2=> Increase the annealing temperature. Choose the good annealing temp by doing a gradient PCR.
**Sol.2 => Increase the annealing temperature. Choose the good annealing temp by doing a gradient PCR.
*The original templates (insert) have contaminations.
*The original templates (insert) have contaminations.
**The original inserts of these libraries is PCR product, probably the inserts originally contain the contamination.
**The original inserts of these libraries is PCR product, probably the inserts originally contain the contamination.
**Since I am using the primers that can bind on adaptors, any thing ligated by the adaptor could be amplified.
**Since I am using the primers that can bind on adaptors, any thing ligated by the adaptor could be amplified.
**'''I assume that Sanger sequencing using Lib. amplification primer is more difficult then using gene specific primers, in terms of getting good quality of sequencing results'''.
**'''I assume that Sanger sequencing using Lib. amplification primer is more difficult then using gene specific primers, in terms of getting good quality of sequencing results'''.
==Optimizing annealing tempurature==
*Test possible annealing temputrature beteen 50C~70C using gradient PCR program ('''Program: G-PCR''')
                1rxn    8.5rxn x 2
H2O            10.0    85.0
DNA            0.5      4.25
Primer-f(10uM)  1.0      8.50 ----- "short-PCR primer" / "very-short PCR primer"
Primer-r(10uM)  1.0      8.50 ----- "short-PCR primer" / "very-short PCR primer" 
2X Fusion      12.5    105.25
-----------------------------
                25.0    212.5 / 8.5=25

Revision as of 06:54, 17 April 2010

Sanger sequencing troble shooting - mp1-mp8 amplicons (PCR products from 04-04-'10)

Background

  • The the 2nd Sanger seqeuencing was performed with special "very-short PCR primer"(The primer modifie from regular library amplification primer with no extension tail and only 21nt => Tm=59).
  • The Sanger sequencing results still showed a very low sequence quality due to the unspecific amplicons.

Possible reasons and improvements (Discussed with technical support):

  • There are unspecific amplification. Unspecific amplication were difficul to be seen and removed by E-gel and still could be amplified in the 2nd round PCR.
    • Sol.1 => Use the old school trick to do gel cutting on target band for sequencing.
    • Sol.2 => Increase the annealing temperature. Choose the good annealing temp by doing a gradient PCR.
  • The original templates (insert) have contaminations.
    • The original inserts of these libraries is PCR product, probably the inserts originally contain the contamination.
    • Since I am using the primers that can bind on adaptors, any thing ligated by the adaptor could be amplified.
    • I assume that Sanger sequencing using Lib. amplification primer is more difficult then using gene specific primers, in terms of getting good quality of sequencing results.

Optimizing annealing tempurature

  • Test possible annealing temputrature beteen 50C~70C using gradient PCR program (Program: G-PCR)
               1rxn     8.5rxn x 2
H2O            10.0     85.0
DNA             0.5      4.25
Primer-f(10uM)  1.0      8.50 ----- "short-PCR primer" / "very-short PCR primer"
Primer-r(10uM)  1.0      8.50 ----- "short-PCR primer" / "very-short PCR primer"  
2X Fusion      12.5    105.25
-----------------------------
               25.0    212.5 / 8.5=25