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Run PCR amplifications in agarose gels


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#1 criscastells

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Posted 03 May 2010 - 06:26 AM

Hi everyone,

I am relatively new in biotopics and you will probably think that my question is so basic, but I would like that someone support my suspicion.

I ran a PCR with primers used in a paper with good results. I tried to repeat their experiment with another cells but I cannot obtain the expected fragment. Instead of the 214bp fragment I obtain a band of approximately 100bp. I know that they are not dimers since they do not appear in the blank (master mix without DNA) and I have studied the secondary structures using bioinformatics tools. So I think that they are unspecific amplifications. After this I want to ask you if there exist some way to study if they really are unespecificities. I have tried to run a BLAST but I obtain no satisfactory results.

Thanks you very much for your help.


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#2 leelee

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Posted 03 May 2010 - 08:57 PM

What do you mean you have studied the secondary structures?

Why do you want to know what the non-specific product is anyway?

I would just work on optimising your PCR with the new sample.

#3 Trof

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Posted 04 May 2010 - 06:11 AM

I ran a PCR with primers used in a paper with good results. I tried to repeat their experiment with another cells but I cannot obtain the expected fragment. Instead of the 214bp fragment I obtain a band of approximately 100bp. I know that they are not dimers since they do not appear in the blank (master mix without DNA) and I have studied the secondary structures using bioinformatics tools. So I think that they are unspecific amplifications. After this I want to ask you if there exist some way to study if they really are unespecificities. I have tried to run a BLAST but I obtain no satisfactory results.

What are the primer sequences and species?

You can always sequence your product using your PCR primers to see what it is.
Is it possible, that your cells have a deletion in the region you amplify? How did you know that your fragment should be 214 bp, did you tested your primers on primer3 or something?

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#4 criscastells

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Posted 07 May 2010 - 05:11 AM

When I talk about secondary structures I mean that I have calculate the free energy formation of primer dimers, and they show that there is not dimer formation.
I use primer blast and it corroborate that the fragment amplified must to be of 214bp. I really don't know if it is possible to be a deletion but I think not since they are human dermal normal fibroblast without any special treatment. Anyway I think that my following step will be to optimize the PCR program.




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