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Queries regarding PCR


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#1 Tamil R

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Posted 19 August 2014 - 11:56 PM

Dear all,

 

I have designed my forward and reverse primer with restriction site (BAMHI) at its 5 end using perl primer. i have run the PCR with an annealing temperature related to the melting temp of the primer complementary to the template. After run the gel, i got a single band around 500bp length but my target gene is 1200bp. after sequencing, i did not get the proper peaks for my product and i got only noise peaks. pl. anyone suggest whether changing the annealing temperature will end up in getting the targeted product or else this primer does not work out. 



#2 bob1

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Posted 20 August 2014 - 12:21 AM

The fact that you got a band that is so different to the one you expect implies that this is a non-specific product. Make sure that the primers have the BamHI site at the 5' end of the sequence in both cases. Also note that the annealing temperature calculations should not include any part of the primers that are not part of the target sequence (i.e. don't include the BamHI site and any other bases that are non-specific to the target)

 

Depending on the target sequence, you may need to play with annealing temp, secondary structure inhibitors, Mg2+ content (polymerase dependent and dNTP dependent too).



#3 Tamil R

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Posted 20 August 2014 - 12:49 AM

Dear bob1, Thanks for your reply. I check out that i have inserted BAMHI site at the 5 end of both forward and reverse primer. Shall i proceed with thermal gradient PCr to carry out at different annealing temperatures? Is the possibility of ending up with my results? 



#4 bob1

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Posted 20 August 2014 - 12:52 PM

I would first check that there aren't some primer binding sites elsewhere in your template DNA - Primer BLAST (on the NCBI website) can do this for you.






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