bisulphite treatment by kit - need information about it and successfull in DNA treatment (Nov/09/2006 )

I think you are referring to the Petronis Group and their publication in Biotechniques very recently.

Whole genome amplification of sodium bisulfite-treated DNA allows the accurate estimate of methylated cytosine density in limited DNA resources
Jonathan Mill, Simin Yazdanpanah, Eva Gückel, Sigrid Ziegler, Zachary Kaminsky, and Arturas Petronis
BioTechniques Vol. 41, No. 5: pp 603-607 (Nov 2006)




can't say I have compared the epitect kit with methyleasy. sorry, but methyleasy is still the one to not degrade your DNA sample which homebrew methods and other kit methods suffer upto 90% loss of DNA.

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Hi everybody!


I tried to use the Cp genome DNA modification kit from chemicon and all my experiments stayed unsuccessfull. Krumelmonster advises me to change the kit.
I have placed an order for a new kit named Methylamp DNA modification kit from Euromedex. Is anyone aware about it?

Thank you for your reply.

Ilofleur

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Hi,

I am a newcomer in this bioforum. I tried both the kits of Epitect kit from Qiagen and the CpGenome modification kit from Chemicon. but after the modification of my genomic DNA, I got nothing by MSP.

I have several questions here, hope someone will help me. this is really frustrating.

1. If the modified single stranded DNA could not check on gel or by spectrometer, then how much modified DNA I should be added in the PCR reaction (for example if I use 50ul of the total volume for the PCR reaction, how much volume I should add as template DNA, because the modified DNA is eluted by the EB buffer in the kit. usually I use 40ul to elute the modified DNA)?

2.If the 3M NaOH shoud be prepare each time you do modification freshly?

3. I used the CpGware software to design my primers, does anyone know if this software is reliable for designing the MSP primers?

Thanks

Pink

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Hi Pink!

I can answer you only for the second question. Yes, the 3M NaOH solution must be prepared freshly each time you do a modification.
I've got the same problem you've got. I've got no products visible in gel after MSP. A member advise me to change the methylation kit. I was working with the chemincon kit. I am to receive a new kit when i 'll try it i will keep you informed.

Take heart! Ilofleur

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Hi Pink, I use the same Kit but use DNAse free H2O to elute (in 30µl). I use between 4 and 8 µl as template in a 50µl reaction mix. That normally works well.
Sorry, never tried it myself. There are a lot of threads in this forum concerning primer design for MSP - maybe you can find an answer there?

Good luck and remember - frustration is 80% of our business

Krümel

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Thanks for your help, Ilofleur. Hope you will work well with the new kit.

Pink

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[/quote]Hi Pink, I use the same Kit but use DNAse free H2O to elute (in 30µl). I use between 4 and 8 µl as template in a 50µl reaction mix. That normally works well.

There are a lot of threads in this forum concerning primer design for MSP - maybe you can find an answer there?

Good luck and remember - frustration is 80% of our business

Krümel
[/quote]


Thank you Krümel, I will try it again.

Pink

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I've recently switched to Chemicon modification kit from the Zymo kit.
However, we kept getting fuzzy bands after PCR.

We are suspecting something wrong with the BS treatment
NaOH is always prepared fresh and timing of incubations,etc are always kept to the protocol.

Does any one having suggestions?

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Hi sharonpek,

I maked the experience that amplification is also dependent from the quality of the Taq you use...
First I used a common Taq, no bands at all, then I compared several mastermixes, and a mastermix for Real-time (Biotools, Nr 10.601) worked fantastic!

Tharom

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We used BD Advantage.
The surprising thing is that the same protocol has worked previously but not now anymore, regardless of the Taq we are using.
The last thing I hope has gone wrong is the Chemicon reagents.

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