LM-PCR yield (Help please!) - (Nov/29/2005 )

What typical yield you got after the first 22 cycles?

I used the Nimblegen protocol. I started with 20ng digested genomic DNA (for the control) and only got less than 500ng total. It doesn't seem working.



Thanks!

digested genomic DNA: do you mean MNase digested? So, if I understand correctly, you are not using sonicated DNA... I've also had (and still have) problems with that protocol. Are you doing the hotstart as indicated? using the same enzymes and conditions? I've read in the TLAD protocol that the nucleotides get degraded after a few freeze-thaw cycles (2-3) so you might want to try fresh nucleotides.

I guess the linker ligation is also quite important since a bad ligation will leave only a small portion of the DNA fragments amplifiable. Do you get better yield with your immunoprecipitation sample ?

Controller

I used genomic DNA after DraIII digestion. Started with blunting the DNA using T4 DNA pol. So it is clearly the LM-PCR did not work. The protocol seems very easy, I have no idea where could go wrong.