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non specific pcr products


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#1 minty

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Posted 09 May 2009 - 12:49 AM

hi. am quite very new with all these ...i need ur suggestions plz....
i had a set of primers with tm 61.9 and 67.2 ..the expected product size is 1.2 kb
am using mosquito genomic DNA as my template
and am getting three bands of 980 bp, 750 bp and 40o bp and a very faint smear background at annealing of 50.6
the bands are also not too bright except the band of size 400bp
how can i get my product?????
sorry for the ignorance but do touchdown pcr can work ????
and how can i set it for my pcr conditions..........
plz guide ...

thanks

#2 T C

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Posted 09 May 2009 - 02:14 AM

I would increase the annealing to 55 or 57 and check once.

hi. am quite very new with all these ...i need ur suggestions plz....
i had a set of primers with tm 61.9 and 67.2 ..the expected product size is 1.2 kb
am using mosquito genomic DNA as my template
and am getting three bands of 980 bp, 750 bp and 40o bp and a very faint smear background at annealing of 50.6
the bands are also not too bright except the band of size 400bp
how can i get my product?????
sorry for the ignorance but do touchdown pcr can work ????
and how can i set it for my pcr conditions..........
plz guide ...

thanks



#3 minty

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Posted 09 May 2009 - 02:40 AM

I would increase the annealing to 55 or 57 and check once.

hi. am quite very new with all these ...i need ur suggestions plz....
i had a set of primers with tm 61.9 and 67.2 ..the expected product size is 1.2 kb
am using mosquito genomic DNA as my template
and am getting three bands of 980 bp, 750 bp and 40o bp and a very faint smear background at annealing of 50.6
the bands are also not too bright except the band of size 400bp
how can i get my product?????
sorry for the ignorance but do touchdown pcr can work ????
and how can i set it for my pcr conditions..........
plz guide ...

thanks


at 56 there is no ampification........

#4 Vini

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Posted 10 May 2009 - 09:21 PM

I would increase the annealing to 55 or 57 and check once.

hi. am quite very new with all these ...i need ur suggestions plz....
i had a set of primers with tm 61.9 and 67.2 ..the expected product size is 1.2 kb
am using mosquito genomic DNA as my template
and am getting three bands of 980 bp, 750 bp and 40o bp and a very faint smear background at annealing of 50.6
the bands are also not too bright except the band of size 400bp
how can i get my product?????
sorry for the ignorance but do touchdown pcr can work ????
and how can i set it for my pcr conditions..........
plz guide ...

thanks


at 56 there is no ampification........



Hi,
I normally like to keep the Tm of the 2 primers close to each other. Another factor to be kept in mind while deciding the annealing temperature is the GC/AT richness of the genome that you are working with. It seems that u r not getting even non-specific amplification at 56. In that case why dont u use an annealing range of 52-55 on a gradient setup?

#5 minty

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Posted 10 May 2009 - 09:25 PM

I would increase the annealing to 55 or 57 and check once.

hi. am quite very new with all these ...i need ur suggestions plz....
i had a set of primers with tm 61.9 and 67.2 ..the expected product size is 1.2 kb
am using mosquito genomic DNA as my template
and am getting three bands of 980 bp, 750 bp and 40o bp and a very faint smear background at annealing of 50.6
the bands are also not too bright except the band of size 400bp
how can i get my product?????
sorry for the ignorance but do touchdown pcr can work ????
and how can i set it for my pcr conditions..........
plz guide ...

thanks


at 56 there is no ampification........



Hi,
I normally like to keep the Tm of the 2 primers close to each other. Another factor to be kept in mind while deciding the annealing temperature is the GC/AT richness of the genome that you are working with. It seems that u r not getting even non-specific amplification at 56. In that case why dont u use an annealing range of 52-55 on a gradient setup?


yah i myself decided to do the same .........
m working on it too ..
lets hope it works..




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