Troubleshooting PCR smear - Urgent,Need help! (Mar/02/2006 )

Hi every body!

Recently, I encounter a problem.

I succefully amplifed a 4.3kb fragment by RT-PCR. I repeat the PCR many time with the PCR product to entrich the fragment about 2 months ago, Every time PCR works. And then I purify the PCR product from the Agarose Gel. Now I try to PCR it again. a long smear from the well to the gel bottom appeared. I test the template on the agrose Gel, results show that the template is still intact, at least doesn't degraded completely. Its concentration is 11ng/ul, Then I dilute the template 10 and 100 times, then with these as the template. Smear goes on. I use the newly diluted primers(which is stored in Sterile TE buffer in a concentration 25uM, I dilute it with ddH2O to 10uM) and new dNTPs, but nothing except an about 1000bp unspecific bands appeared. Changing Mg2+ from 1.5mM to 1.6mM, It still does not wok!

RXNS system:

(10mM)dNTPS:0.4ul, FW primer and RV primer:0.5um(final Conc.) each, DMSO:5% final CONC. Template DNA 2ng/ 0.2ng each tube. 10X Buffer 2ul, DyNAzyme EXT DNA polymerase(FrOM NEB), add ddH2O to 20ul.

RXNS program:
94C 3min

94C 25sec
60C 30sec
72C 4min
for 35cyces

4C hold

Anybody can give me detailed suggestion! it is rather urgent!

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have you ordered an entirely new batch of primers?? could be primer degradation....results in all sorts of random priming events and gives a smear every time...this happens even if you store them in the freezer, after many months

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Hi Aimikins:

Thanks for your reply!

You know I did not solute the Primers into the ddH2O, instead, I soluted it in TE(PH8.0 buffer, sterile) to a concentration 25mM and stored it in-70C. when using, I dilute it to 10mM with sterile ddH2O. You say the oligonucleotide could yet degraded in PH8.0 TE Buffer?

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sure, eventually...although it happens faster with water, I think

how long have you been storing them at -70? I have known many people that say you can store them for years. in my personal experience, I find that after around 6 months, sometimes you will begin to see signs of degradation

another question, sort of from left field...are you sure your diluent water has not somehow become contaminated with sufficient nucleases to kill the primers?

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Hi aimikins:

Thanks for your reply, I Later dilute the 50uM primers with steriled dd-water, which I autoclaved the water at 121C, for 15min. So I am sure they were not contaminated with nucleases. However, nothing appeared on the Gel after the PCR amplification.

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