i am trying amplification of a gene from genomic DNA, as my one of the primer had secondary structure i used high annealing temp and now could get pcr product but is not enough so i tried re-amplifying the pcr product with the primers, but it is not giving the product at all, why could this happen? any suggestions to solve this problem?
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#2
Rosario
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Posted 10 February 2012 - 05:55 AM
Hi,
In my experience is already happened that the secondary PCR performed on the product of the first one with the same primers does not work well. Sometimes I got smear and sometimes no bands completely.
In my opinion sometimes can occur, during PCR, a short deletion at the ends of the product and this does not allow the annealing of the same primers again.
Why do not you try to increase the cycles number (5 are enough) during the first PCR to get more DNA product??
In my experience is already happened that the secondary PCR performed on the product of the first one with the same primers does not work well. Sometimes I got smear and sometimes no bands completely.
In my opinion sometimes can occur, during PCR, a short deletion at the ends of the product and this does not allow the annealing of the same primers again.
Why do not you try to increase the cycles number (5 are enough) during the first PCR to get more DNA product??
#3
Trof
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Brain on a stick
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Posted 10 February 2012 - 01:36 PM
Try DMSO (different concentrations 1%, 5%, 10%) with the lower temperature to decrease the formation of secondary structures.
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#4
Evanescence
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Posted 17 February 2012 - 05:39 AM
I agree with "Brain on a stick" i have same problem before but when i used DMSO with lower ann.temp i got nice band
but with double band, and re amplify again , got easier this time and time and purify( if you have multiple band)...
could be your gene has high GC content? or your primer responsible for that...secondary annealing maybe?
but DMSO can help to solve this
yeah!
but with double band, and re amplify again , got easier this time and time and purify( if you have multiple band)...
could be your gene has high GC content? or your primer responsible for that...secondary annealing maybe?
but DMSO can help to solve this
yeah!
