nested PCR - (Sep/24/2005 )

Dear Veterans,
I did a nested PCR for a large fragment (8Kb)cDNA. No amplification found in the first round. There was an amplification in the second round, though it matched the size of the first round product. And there was smearing near the well.
Q- Is the smearing due to the long fragment size?
- Shud i try with long template PCR?
- Is it possible to get the first round product with the second round (inner) primers?

Thanks for ur time.

What RT are you using? Which is the enzyme you're using for your PCR? How much smaller is the fragment you should get with your inner PCR when compared to the fragment you expect with your first PCR?

As you are getting a fragment in the second PCR, I don't think long template PCR will be necessary.

Smearing can have loads of reasons, consider changing the amount of Mg, the annealing temperature, the cycle number of your second PCR... (Just yesterday I did a PCR of 8,15 kb and got a nice single product on a gel, without any smear, so the long fragment size in itself should not be causing the smear).

Dear Ezhil,

Smears around the well are usually high molecular weight DNA, most likely are your genomic DNA.
How pure is your extracted RNA? A260:A280 ratio?

I would like to suggest you to increase your salt concentration, by using:
i) 1.2 x, or 1.5 x PCR buffer (KCl) and
ii) 2.0, 2.5, 3.0mM Mg.

Increasing KCl generally will make your primer bind batter to your template. Increasing Mg concentration will make your primer extension perform faster. Hence PCR efficiency will by increase and you get more product. Probably you can get a band after you first PCR.

However, increasing salt concentration will also decrease PCR stringency. Thus be aware of unspecific band. So you have to do a little optimization


Best regards

Thank you for your answer. sorry for the delayyyyed reply.