Question on PCR - How to add sequence fragments to DNA ? (May/13/2005 )

Hello, I was wondering if anyone could help me. Im trying to add two fragments to a DNA sequence, one to each end of the fragment by PCR. My primers have complementary seq to my target and the piece I want to incorporate, but I'm not gettign any product. Does anybody now anything that could help me?

thank you

how long are your tails? and what is the Tm of the part of the primer that is complimentary to the target seqeunce?

I used to add tails to my primers by T4 RNA ligase and then perform the PCR with the tailed primer and then a second round of PCR with the tails by themselves.

hope this helps

Nick

Hello,, thank you for responding to my question. My primers are 61 and 66bp longs (this includes the tails) the Tm of the whole primers is 70.5 and 70.3,, but the Tm for the fragments complementary to the target are ~61 and ~60.

Hello, I was wondering if anyone could help me. Im trying to add two fragments to a DNA sequence, one to each end of the fragment by PCR. My primers have complementary seq to my target and the piece I want to incorporate, but I'm not gettign any product. Does anybody now anything that could help me? 

thank you

hi,

what is the Tm you are using for your PCR reactions? I would probably go for 58C as should ensure that your gene specific regions will anneal.

good luck!

Nick

Ok, Thank you!! I've done the PCR at 45 and 60, but nothing; but I can get products if I use one tailed primer only. I'll try 58C.

have a good day
naomi

You didn't get a product at 45C? hmmm have you also performed a positive PCR amplification control with primers to your template without the tails? This would give you an idication of whether your PCR is in fact working (and that your gene specific primers are actaully amplfyingyour gene) and then use your tailed primers.

Nick