PCR troubleshooting - Product streaks and primer dimer formation

[biologyred]

Hi, For that PCR run, some of the sample are are able to be amplified but some of my PCR product result in streaks near the well and some primer dimer formation. I have already tried diluting my template concentration and still does not produce any result, what is the next step for my PCR trouble shooting?

[jiajia1987]

How many cycles did you do? perhaps you might want to reduce the number of cycles to reduce the random-length PCR products.

[biologyred]

The Cycling condition has 35 cycle. It usually works ( already optimized) for other standard samples but some of the samples just wont produce a specific band.

[Aga]

Are you sure the other samples contain the DNA fragment you try to amplify? What about the template itself - is it purified enough or may it contain any PCR inhibitors that other samples were free of?

[biologyred]

I already consider the possiblity of PCR inhibitor, I tried a series of dilution for these samples but it does not work, are there strong possibility that my sample does not have the DNA template that I wanted?

[jonoave]

What template are you using? Is it extracted genomic DNA or reamplification of purified/non-purified PCR product?

Genomic DNA is more stable, and can survive quite a number of freeze/thaw cycles. However, non-purified PCR product is less stable and can degrade in a few days with frequent freeze/thaws. If you're using PCR products as template, my advice is to start from fresh.

[biologyred]

I am doing the nested PCR approach, first with RT-PCR and then normal PCR run.The first PCR run was using samples that was freshly extracted (RNA).

[jonoave]

biologyred:

Your description is slightly confusing. Am I right to assume that what you did was;?
1. RNA extraction
2. RT-PCR as in reverse-transcription, which generates cDNA.
3. normal PCR, using the generated cDNA as template.

The term "nested PCR" is usually used to refer to a subsequent PCR cycle, ie. a second PCR using the PCR product from the first PCR run as template.

Anyway, referring to your problem - what is the difference between the PCR samples that you run where some can be amplified and some couldn't? Are they simply replicates of each other? Or are they using:
1. different template?
2. different primers?
3. different mastermix composition?
4. different annealing temperature?

Try optimising the PCR by identifying what you did diffferently between the samples that were successfully amplified and those that has smears.

[spal]

Were the PCR conditions the same for each of the samples you tried to amplify? If all of the conditions are the same, but the DNA templates are different, a potential problem might be the primers you are using. Are they universal? It could be that there is a mutation at the primer annealing site on some of your template DNA samples.

[Aga]

If you are not sure whether you have your RNA or DNA degraded or extracted at all try to check it spectrophotometrically and/or electrophoretically each time before you start your reaction.
The question is whether you get your cDNA each time? Do you check it after RT?