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