Sam:LabNotes/Microbiome-new/2010-4-16
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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) - program set up (07-01-2009)
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 NOTE: 1. The expected Annealing temp for short-PCR primeris 64. 2. The expected Annealing temp for very-short-PCR primer is 59.
Results
- PCR program (G-PCR):
Step1 94C 2min Step2 94C 40sec Step3 50C-70C 40sec Step4 72C 1min Step5 Goto step2 for 34 cycles Step6 72C 10min Step7 End
File:Sam041610-gradient PCR on short -PCR primer.jpg File:Sam041610-gradient PCR on very-short -PCR primer.jpg