I am running an assay wherein I will amplify an oligo (1.5kb) - but in my experimental group I am expecting the oligo to break during replication. If I load 2ng of DNA and then run 40 cycles of PCR, will that generate enough DNA for me to run on a gel and visualize even if I get strand breaks and fragmentation? (fragments in the range of 100-300 bp)
Yes, if it works you should get more than adequate resolution
you do not say what proportion of thr 2ng is your pcr target but if it is all amplifiable then simplistically 40 cycles is one million million times more product than you started with, This is about 2000 grammes of product in a linear pcr so I feel that Nathanial is right even though pcr is not linear over so many cycles
Hi Damian,
just to clafify, you expect the oligo to break during amplification? (opposed to input that is either fragmented or not?)
you should keep in mind that after breaking, there is no exponential amplification anymore, so there will be a gygantic difference in abundance between the fragments and the complete products.
For example, assuming your break occurs in the first cycle:
- the fragment will be amplified 40x (no exponential amplification, so only amplification from one side, once each cycle is 40 fragments in total
- the complete product will be amplified 2^40 times ( about 1.1*10^12)
so if you were to run this on gel, the signal intensity would be so extremely different, that you'd never actually see the fragments
I'm actually assuming that you have input which is either broken or not (due to a translocation or deletion event?)
if this is the case, and you know roughly where the deletion occurs, it makes more sense to make a PCR traversing the deletion site, where the fragment size changes if the deletion has taken place. (ideally you'd also be able to amplify the undeleted DNA, but if the deletion is too large, this will be difficult to amplify).
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