Possible to clone when you have only 20ng PCR product? - (Sep/04/2006 )

Hi there,

I was wondering if anyone knows the answer to whether its possible to clone successfully (and get good sequences) starting from very low quantities of PCR product, like around 20 ng of a 600 bp product?

I am trying to sequence a gene that is expressed at really low levels. On the first amplification, the band is only just visible on an EtBr gel. Whenever I try to re-amplify my initial PCR product, it turns into a smear.

Any ideas?

Don't see why it shouldn't be possible, only problem might be to get your DNA concentrated enough to use in ligation reactions. (I usually ligate 10-20 ng of insert of that size).

On the other hand, is there no possibility to increase the yield of your PCR? (Increase number of cycles, decrease annealing temp, increase template a bit maybe) Might be easier to work with afterwards. If you re-PCR, you want to dilute your initial PCR product a lot and you might get a better result.

Thanks Vairus,

I will try it out. Do you clean up your PCR product and then elute in very little ddH20 to get it concentrated enough? Or do you elute in , say, 50 ul and then ethanol precipitate?

I will also try doing the 2nd pcr with diluted product.

thanks!

try to make 5 reactions of the first PCR and pool it and use it to cloning it.

20ng is still sufficient for cloning.

I agree with Vairus. work to increase your product. whether or not the gene expresses at low levels shouldn't matter; you still get the same number of copies in a chromosomal DNA prep

what have you tried to increase your product? and, are you certain your template is good?

are you sure about your first amplification? could it be that you areting smear because of unspecific amplification? I would try to adjust the PCR first and get defined bands before cloning

I would check the amplification conditions to get rid of the smear first

Thanks all,

Aimikins, thanks for that. Its cDNA, not genomic. The template is not perfect. I ran the RNA on a bioanalyser before I started. It wasn't particularly abundant and it was slightly degraded. Its probably impossible to get more template at this stage, so I'm trying to just work with what I have.

I am trying to amplify a gene from several different species, using primers that are slightly degenerate (ie 2 degenerate sites in a 20 bp primer). Unspecific priming has happened in some of the species. I cut out the bands of interest and clean them up first.

I will try again with the second round of PCR and a new enzyme, and we'll see how it goes.

sorry, smurray, didn't mean to be a PIA

it seems that the consensus is to dilute your template; perhaps try a titration of the first product and see?

anyways, good luck

Re PCR.