bisulfite sequencing cloning problem (methylation of DNa) - bisulfite sequencing cloning problem (methylation of DNa) (Apr/21/2010 )

I'm really desperate because for few months my cloning for bisulfite sequencing experiment is not working.

First we tried to do the cloning with pDrive. We've done the bisulfite treatment of our DNA samples with standard EpiTechBisulfite kit from Qiagen. Then we did PCR with appropriate primers and polimerase Taq and then we isolated DNA products from agarose gels with Qiaquick Gel Extraction kit. The first problem was that we get small amounts of PCR product extracted from gel and additionally it was weird that sometimes one PCR worked and another time the same PCR didn't work, but maybe its because of primers. Then we did ligation and transformation according to Handbook for pDrive cloning. The problem was that there were no colonies on plates and then when we ordered new Ez competent cells from kit we get a lot of colonies but after miniprep it occured that we had just pDrive in bacteria but no insert. I was thinking that maybe the problem is with phosphorylatiom, I mean: I read that when we try to clone insert which we get from pcr we should phosphorylate the insert because during pcr it is dephosphorylated and because we don't cut insert and the pDrive vector (pDrive has a tail of A bases which should link with tail of insert which is added by polymerase and they hould connect according to manufacturer) maybe they can't link with each other.

Now we did another cloning procedure with pUC18. Again we did earlier steps the same. Now we cut the insert and the vector with 2 enzymes. We did cloning and again we didn't get any colony....I don't know what is wrong, we changed ingredients for new ones, we always do fresh plates and we tried different conditions for transformation and ligation and we don't have nothing.

Maybe someone have an idea?

mon_gronek

do proper controls! This will tell you what is going wrong during your cloning procedure.

for details check this:
http://bitesizebio.com/2007/11/20/pin-poin...ation-problems/

Controls are essential! Do NOT skip them!

Regards,
p

pDNA on Apr 21 2010, 05:23 PM said:

did you check the polymerase you are using produces A overhangs?

doesn't explain your restriction digestion and cloning, where did you place your restriction sites within the primer? there has to be some overhanging sequence flanking the site to ensure the enzyme cuts.

As pDNA says, controls are essential here.

Nick