dna degradation

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problematic pcr
problematic pcr's picture
dna degradation

can u all tell me the reason that i'm not getting the bands in the dna samples now in which i've already got results.is my dna degraded?well i'm the getting the bands in the positive control but smearing still persists. 2ndly plz tellme triton-x in buffer affects the reaction or not and if yes than in what way?

plz ans as soon as possible.
thanks.aur mailme to eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%6b%68%75%73%68%5f%6e%61%31%37%40%79%61%68%6f%6f%2e%63%6f%2e%69%6e%22%3e%6b%68%75%73%68%5f%6e%61%31%37%40%79%61%68%6f%6f%2e%63%6f%2e%69%6e%3c%2f%61%3e%27%29%3b'))

Jason King
Jason King's picture
Is the Triton in there

Is the Triton in there because you have made cell extracts? Usually there would be a further purification step (phenol:chloroform then ethanol precipitation or an affinity column purification) to get pure(er) nucleic acids to use as a templete in a PCR. Triton should then not be present.

Often people use a nice clean plasmid DNA as a positive control for samples that have been generated via RNA prep + cDNA synthesis. This is not a good idea. The positive control should have been through the same steps as the samples it is controlling for.

DNA will degrade too, especially if the solution it is in is not clean (pure DNA) or if it is not kept frozen and in pH 7-8 buffer.

Dilip Fernando
Dilip Fernando's picture
Check the DNA storing

Check the DNA storing conditions too
In TE buffer, for long term storage

Gel conditions too might affect the DNA migration so check that too