Standard PCR Troubleshooting and Optimization
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Standard PCR Troubleshooting and Optimization
A general guideline for PCR optimization including reaction conditions and components (from Applied biosystems).
Some considerations for standard PCR:
1. Sample Volume and Reaction Tubes
2. Template DNA or RNA
3. Primers
4. Deoxynucleoside Triphosphates (dNTPs)
5. PCR Buffers
6. Magnesium Ion concentration
7. DNA polymerase used
Last update 25-Feb-2006, Rating Poor of 4 votes.
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Hi!
I am working on standard pcr of Bacterial leaf blight primer RG 136, IN RICE genomic DNA. Previously, i was getting good amplification with the other primers, but with this primer it is showing very faint amplification , bands are very weak, & sometimes, unamplified DNA is seen near the wells of the gel. there's no problem with the primer functioning, as it has been checked earlier! kindly suggest what cud b the reason for this!
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hello
I am doing my RT-PCR reaction from long time and had no problems at all. But now a days my product is thinner then earlier(as earlier I used to get the brighter and thicker band of RT-PCR product). My product size is 1.1kb.
I checked for all parameters including enzymes buffer and primers and everything is working properly as when I am putting same reaction in two half that is instead of amplifying 1.1kb I split and amplify whole region using 4 primers (app. 600bp two products) results are good that is I get brighter and thicker band.
I am using the product for seqencing so I need good and brighter band otherwise I get bad sequences.
Can you plz comment where the problem lies.
I think it may be RNA giving trouble.
I want an expertise comment is that if RNA is less there is problem with amplifying long regions but small regions get amplified well]
Its urgent if u can solve the problem.
Thanks
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hi
i am working on methylation specific PCR of p16. but unfortunately i am getting smear at the site where band shoul be present. If any body has solution and suggestion to overcome the problem, please express. I have used all possible concentration of MgCl2 (i.e from 1.5mM to 14mM) and different concn. of dNTP and Taq. still not getting the result.
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very easily explaned molecular biology techniques also pcr trouble shooting
Rating: Very Good
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