Troubleshoot PCR smear problem - all I got was smear!! (Aug/18/2004 )

[b]primer stocks DO DEGRADE - we had problems with primers ordered from Invitrogen - it was a big problem in the lab because simultaniously 4-5 fifferent PCR reactions did not work so I measured primer mass concetration on Bechman DU spectrophotometer and it turned out that molar concentrations were not 40 microM - instead they were 20 microM - and really wqith degraded primers after electrophoresis under UV you see smera but with new aliquote of primers PCR bands are very sharp and clear.
pozdrav!
Ivana

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True, true! Pozdrav Ivani:)!

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hi evry body
well i am working on the water buffalo TSH beta subunit .The region i m going to amplify is about 492 bases long .I have designed forward primer n reverse primer of length 20 n 25 respectively. after Pcr i got only a smear but much sharp around 100 bases lower than my required product.Is it a non specific amplification . i have used sequence specific primers for cDNA synthesis.For agarose gel i have used TAE buffer. So where is the culprit??????????????
thnx in advance

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I encountered the bright smear / faint band problem yesterday!

I had initially thought that I loaded too much template. I did first-stranded cDNA synthesis (ImPromII) using 1ug of total RNA extracted from 1e6 CHO cells (RNeasy), giving me 20 ul of cDNA product (I spec-ed the DNA but it gave me weird numbers). Anyhow, I loaded 5 ul of template in a 50ul reaction using Invitrogen's PFX. Then the problem appeared.

Suspecting that I loaded too much template, I decreased the loading to 2ul and raised the annealing temperature by 5 degrees C. My gel is running now but I suspect the problem has not been solved (cos I took a peep after running the gel for 10 minutes).

What other things could I have missed? I'll try to extract the cDNA again and repeat PCR using a different set of reagents. I'm reasonably confident of the primers... What other factors can I vary?

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I used to work in a lab next to a facility that actually produced oligonucleotides/primers. They said it was best to store working aliqouts of primers at 4 degrees. The freeze thaw causes degradation very easily to primers. Primer stocks should be stored at -20 degrees. [







quote name='aimikins' date='Mar 24 2005, 06:22 PM' post='12964']
It seems that this topic has been answered to death, but I had a thought that no one else brought up, and it may help someone in the future?

Oligos will degrade over time, even when properly resuspended in nuclease free water or buffer and aliquoted and stored at -80...eventually they will degrade and this could cause non-specific priming. Based on my personal experiences in the lab, I would consider this problem first if I began to see smeary gels, particularly if I were careful about good technique everywhere, especially if the problem seems to get worse over time.

I had a similar experience once and spent a few months going through every detail of my protocol...remade and reordered my other solutions 8 trillion times and was very frustrated...and it just turned out to be oligo degradation.
[/quote]

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I had a problem about smearing whenever I ran the gel. I worked on bacteria DNA. Have you purified your DNA? If you have time, treat with RNase and do again. It helped me much better..Good luck

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Hi Stauffer,

I would like to treat my pufified DNA with RNAseA (to prevent smears but even more to determine the exact DNA concentration of my sample). I assume, I need to repurify the DNA after RNAse treatment for susequent PCR amplification. Could You share your protocol with me for RNAse digestion (RNAse kind, temperature, duration, concentration, buffer) and for purification of DNA after treatment ?

All hopeful

Barbara

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According to the back of the pfuUltra trouble shooting guide (Stratagene), a smear could be caused by too much polymerase or too long of extension times. I have not varied the polymerase concentration much, but decreasing extremely long extension times has helped my smearing issue.

-My two cents,
Leigh

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Even when primers look beautiful & perfect, sometimes they just don't work, or simply do not give reproducible results. Trying to figure out why is often a pure waste of time.
Get new primers, it's the cheapest and fastest way to get around it.
When primers work ok, it's a whole different story!

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Dear Kunda
Have you tried decreasing the Taq amount? Sometimes it can cause smearing too .

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