5 replies to this topic

[klamc]

Hello,

I will start doing BS PCR and cloning into pGEM vector since our several atempts to do pyrosequencing are not working. For some reason, the PCR product that we sent is never of good quality,

So my questions are:
1. Is it ok to try to amplify and clone a 3kb BS treated DNA fragment?

2. Im not familiar with the protocol, im not sure if I got it right, but as far as i can understand, I do BS treatment to gDNA, then amplify region of interest with appropiate primers, and then do the cloning into TOPO or pGEM, then pick at least 20 (?) transformed colonies and send that to sequence and then calculate the %of methylation. But is not clear to me if I have to make 20 different PCR reactions and then transform and pick one colony of each or I just do 1 PCR reaction and pick 20 colonies out of this.

Thanks!

PS: if there is a paper that explains this more clearly, please let me know... thanks again

[pcrman]

Hi Klamc,
>Is it ok to try to amplify and clone a 3kb BS treated DNA fragment?
3kb is too big to amplify. Amplicon size should be <500 bp.

> Im not familiar with the protocol, im not sure if I got it right, but as far as i can understand, I do BS treatment to gDNA, then amplify region of interest with appropiate primers, and then do the cloning into TOPO or pGEM, then pick at least 20 (?) transformed colonies and send that to sequence and then calculate the %of methylation. But is not clear to me if I have to make 20 different PCR reactions and then transform and pick one colony of each or I just do 1 PCR reaction and pick 20 colonies out of this.

Yes, sequencing 20 colonies is good enough. You just need one PCR reaction and clone the product into a vector.

How does you PCR work for you so far? Did you see good PCR bands?

[vilperte]

Hello!

I've some basic papers about methylation.
You can download some from these links: http://www.springerp...8-1-59745-522-0 or http://link.springer...-31390-7/page/1
I also have some others that I don't remember where I got. I don't know how to attach PDF files here (is it possible?) but if you want I can send you by e-mail.

[klamc]

Thanks to both of you! I haven't tried the PCR yet, only designed the primers. I just wanted to make sure I was am in the right way

[klamc]

pcrman, on 15 February 2013 - 08:13 PM, said:

Hi Klamc,
>Is it ok to try to amplify and clone a 3kb BS treated DNA fragment?
3kb is too big to amplify. Amplicon size should be <500 bp.

> Im not familiar with the protocol, im not sure if I got it right, but as far as i can understand, I do BS treatment to gDNA, then amplify region of interest with appropiate primers, and then do the cloning into TOPO or pGEM, then pick at least 20 (?) transformed colonies and send that to sequence and then calculate the %of methylation. But is not clear to me if I have to make 20 different PCR reactions and then transform and pick one colony of each or I just do 1 PCR reaction and pick 20 colonies out of this.

Yes, sequencing 20 colonies is good enough. You just need one PCR reaction and clone the product into a vector.

How does you PCR work for you so far? Did you see good PCR bands?

But what if I can get the 3kb pcr to work(using a HF polymerase) and then sequence with internal primers?

[pcrman]

Quote

But what if I can get the 3kb pcr to work(using a HF polymerase) and then sequence with internal primers?

Then, most likely the product is not from bisuilfite modified DNA, but from unmodified or incomplete modified DNA.