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DNA amount for making Lib-L from PCR fragment
Hi all,
I am going to make ligation library from PCR fragments.
Roche recommend to use 0.5 microgram of DNA (10^11 molecules as 500 bp) for this and after purification it becomes around 10^9 ends of DNA fragment.
It might mean Tags are ligated only to a few % of ends, and thus only 1/10^4 molecules have Tag at both ends.
In the protocol, 10^9 ends looks like to mean 10^9 fragments having Tags at both ands and thus can be used for emPCR.
So, I must be missing some point, but what is that?
I am going to make ligation library from PCR fragments.
Roche recommend to use 0.5 microgram of DNA (10^11 molecules as 500 bp) for this and after purification it becomes around 10^9 ends of DNA fragment.
It might mean Tags are ligated only to a few % of ends, and thus only 1/10^4 molecules have Tag at both ends.
In the protocol, 10^9 ends looks like to mean 10^9 fragments having Tags at both ands and thus can be used for emPCR.
So, I must be missing some point, but what is that?