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Bisulfite modification kits and PCR


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5 replies to this topic

#1 Kiwicloner

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Posted 19 July 2004 - 03:56 PM

I was wondering if anyone could provide an opinion on the difference in PCR amplification effiency from DNA that has been Bisulphite modified using either the MethylEasy Kit or the EZ DNA methylation kit.

We are trying to amplify a PCR product of about 400 bp for cloning and we also need to use the template for MSP.

Has anyone found that DNA is more easily amplified from one of these kits compared to the other?

Thanks for your help!

#2 pcrman

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Posted 19 July 2004 - 05:31 PM

I don't think the kits from different vendors will make much difference, although I've never used any of those (I usually use the kit from chemicon, originally Intergen).

If your DNA is from cell line or fresh tissue, the chances of getting 400 bp amplification are high and the modified DNA should be also OK for MSP.

For modification, use 1-2 ug DNA and elute in 30 ul water at the last step. Use 2 ul for a 20 ul PCR reaction.

If you are amplifying DNA for sequencing or cloning, start at 40-45 cycles and hot start your PCR or better use some hotstart taq such as the JumpStart from Sigma which really makes a big difference.

Good luck.

#3 methylman

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Posted 20 July 2004 - 12:19 AM

I have amplified a 1400bp region using the Methyleasy kit. I find that we don't lose a lot of DNA with that kit. I have used the EZ kit, but if you follow their instructions you only get enough for 5 PCRs or so (we got more than 50 with Methyleasy).

I think it is important to have clean DNA otherwise it may inhibit the PCR later. Do you rountinely Phenol/Chloroform extract? It helps a lot.

#4 pcrman

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Posted 20 July 2004 - 08:00 AM

Do you rountinely Phenol/Chloroform extract? It helps a lot.

At what stage you do this, before or after modification?

Edit:

OK, I got it from your another post. You mean before modification. Do you think that is crucial, since you have another chance to purify your DNA after modification?

Edited by pcrman, 20 July 2004 - 08:07 AM.


#5 methylman

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Posted 20 July 2004 - 06:27 PM

I think it depends on the method of purification after the modification. Columns might be better to clean up the remaining DNA after modification, but there is a big loss in recovery as well. Methyleasy precipitates the post-modified material which will give you a better recovery, but may also bring down impurities in the original DNA prep. As I want as much modified DNA as possible I start with really clean DNA and use Methyleasy.

#6 pcrman

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Posted 10 August 2004 - 09:26 PM

start with really clean DNA

It seems true. Recently I modified two batches of DNA at the same time with one was not very clean (recoved from luciferase assay). The result was the "dirty" DNA was not completely modified as shown by sequencing.




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