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