Hi,
i am currently looking for an experiment alternative to southern blot. I heard that pcr based assays give results quickly ( let's say within few hours). I want to perform sybr green relied pcr to detect copy number of my interested gene in the human genome like in southern blot. however, I couldn't find a proper protocol. If you address a protocol or share your experience, I will appreciate.
Thanks.
Dear Mert
techniques like Southern blotting which take 2 days have fallen out of favour with the advent of techniques like qPCR that can be carried out in under 1 day
In essence standard curves comparing Ct with copy number or conc of nucleic acid are used to determine unknown copy number/concentration of a sample with a known Ct
See for example:
https://www.agilent.com/cs/library/slidepresentation/Public/Quantification_of_qPCR.pdf
all depends what you ould like to check. Even if PCR is faster and simpler, for some cases, southern remains the best.
Agree with the author above. If there are multiple copies of GOI scattered around genome, Southern blot may not resolve all of them, and qPCR may also be inconclusive. You may need to do NGS using long read platform to convulsively determine the copy number. qPCR is handy for an estimate.
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