Sam:LabNotes/Microbiome-new/2010-4-16

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Revision as of 23:40, 16 April 2010 by >Sam Chiang (Created page with '='''Sanger sequencing troble shooting - mp1-mp8 amplicons (04-04-'10)'''= ==Background== *The the 2nd Sanger seqeuencing was performed with special "very-short PCR primer"(The p…')
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Sanger sequencing troble shooting - mp1-mp8 amplicons (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.