Bisulfite genomic sequencing - Bisulfite genomic sequencing (Aug/11/2005 )

Hey. Hope someone can help me with this one. I basically have three problems all related to bisulfite genomic sequencing.

Background: After modification using Chemicon's CpGenome modificatin kit I am able to get a PCR product that matches the expected size. (I seem to get about 1-2ng of PCR product). I also did a restriction enzyme digest to confirm that I was amplifying the right region.

The primers I am using have EcoRI restriction enzyme sites attached to their 5' ends. I am trying to clone these PCR products using pGEM3Zf(+).
So far I have been able to obtain - at most - two clones from a single transformation (my control plates are clear). Direct PCR of the white colonies yields a PCR product that matches the expected product size. However, I have been unable to obtain PCR product from the isolated/purified plasmids - after carrying out phenol:chloroform extractions.

There are three problems:

Problem 1: I don't seem to have enough PCR product for the transformations (I only get two clones); using my 1kb marker as a guide, it seems that I get between 1 and 2ng of PCR product. This may be a problem related to the bisulfite modification.

Problem 2: I only get two clones. This seems to be a problem of too little PCR product. alternatively, perhaps my choice of cloning technique is unsuited to bisulfite genomic sequencing. Everyone else seems to do TA cloning. In contrast, i'm trying to ligate my PCR products (which have EcoRI sites attached to them) into EcoRI sites on pGEM3Z(f+).

Problem 3: inability to amplify my region of interest (i.e. the insert) from phenol:chloroform extracted plasmids. These plasmids are obtained from white colonies on my insert:vector:ligase plates. My control plates are clear. This should mean that the ligation didn't work and that there is no insert to amplify. However, I get amplification when I do PCR directly (I.E. when I poke the white colony with a pipette tip and then poke my PCR mix with the pipette tip).

Can anyone suggest anything?

Oh. and recently I have been getting these weird PCR artifacts, which appear in my positive and negative controls. However, my actual PCR product only appears in the positive control. I am beset with problems

Hi Phillip,

what are you PCR conditions for your Bisulfite PCR prior to cloning? How many cycles are you performing? are you using a nested PCR strategy? If you are only performing 25cycles and one round of PCR, this would explain why you get so little product after the amplification (you could try and drop the Tm in your PCR a degree or two and see if your amplicon comes up).

How are you preparing your PCR amplicon prior to ligation into pGEMT? I am aware that the ligation rection can be sensitive to many things, including the type of water your DNA is resuspended in!

As for your inability to amplify your plasmid, did you perform a restriction digestion and see your insert drop out from the plasmid? The reason why your PCR has not worked is that the polymerase is very sensitive to the presence of phenol, if there is some contamination there, your PCR will fail. A restriction digest and insert dropout will tell you for sure that you have successfully cloned your amplicon.

Hope this helps, all the best, Nick