what can i do with this pcr? - amplification of cDNA , with primers tm 50 and 67 (Jan/13/2007 )

the length of the piece i want is 1300 , i tried many times, but i dont have, i have only unspecific products.
what u suggest to do, to increase mg concentration, ??/

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try increasing annealing temp. to 60 and 65C. Also u could change Mg conc.

Use higher amounts of template.

Good Luck !!!

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your target sequence is GC-rich or repeat ? 1300bp is easy to amplify. you may add DMSO , betaine to your PCR reaction , if your sequence is GC-rich or repeat. you also try Hot-start PCR

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Tell us much more. Template. Primers. Amounts. Cycling conditions. Enzyme. Buffer.

If your first inclination is to increase template, you are probably using way too much. Try serial dilutions of your template 10x, 100x, 1000x. More is worse, typically.

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try grediant PCR or touchdown program

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actually, i made cDNA from virus RNA, and i tried for this piece of cDNA many times to make pcr, with these primers, and always give me unspecific bands, i put 5 microl from cDNA, and 1 micro dntps 10 m mol, and MG is 1.5 m mol.


i tried by different templates originated from different RNA extractions, at many ta like 50,53,57,60,67. and ihave always non specific bands.

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have you tested the cDNA with another set of primers you know that work?
with a tm of 50 (did you mean 60?) and 67, there should be a temp that will work with both primers.
if you're using taq... you have to extend for about 1.5 min. check your cycling conditions.
try a gradient PCR, as suggested earlier.

V

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if your primers Tm is 50 and 67, I would suggest dropping the annealing temperature as low as 45 Celsius.

and there are the suggestions of adding adjunts to your PCR. They could help.

but if everything has been tried... there is the final suggestion of purchasing new primers... ones with a more sensible Tm with a more sensible Tm difference ( 0 - 2 Celsius rather then 17 Celsius)

EDIT: Oh by the way, what polymerase are you using? Taq isn't really the best polymerase with a PCR of this lenght.. 1.3kb is near the 'easy' limits of Taq... around this point you start to need real optimisation to go longer and PCR errors start showing their heads. I would suggest a proof reading polymerase to do this PCR.

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