CpGenome kit not working anymore - (Jul/11/2005 )

I ordered the Chemicon CpGenome kit in May, and was getting great results at first with my modified DNA in MSP. However, about a month ago it stopped working. I cannot get any of my samples to amplify in MSP, even though my previously modified DNA samples amplify the same as before. I contacted Chemicon and they sent me a brand new kit free of charge. I also went out and bought control unmethylated and methylated DNA from Chemicon. I also changed all my reagents (3M NaOH, EtOH, etc). Still it does not work!

I know that the PCR itself is fine, because my previously modified DNA samples still show a band. It is just all of my new samples that I try to modify that do not work--not even the commercial control DNA.

Has anyone else had this problem? Is the kit really sensitive to environmental changes? The weather has been getting hotter the last few weeks. That is the only thing I can think of at this point. I may also look into ordering new NaOH in solution...the pellets have been hanging around for quite awhile so maybe they are not pure?

If anyone can provide any suggestions it would be greatly appreciated.

Hi may, is the bisulfite reagent in a solution?

With homemade bisulfite I know it is light sensitive and also degrades quite quickly and I routinely prepare fresh bisulfite reagents.

Have you changed your DNA isolation protocol in some way? has the proteinase K gone off? improper DNA extraction leads to inefficent bisulfite conversion which could then lead to no amplification using your MSP primers because the samples have not converted properly.


did the controls from chemicon work? It does sound like a bisulfite reaction failure.

Good luck

nick

Hi nick, thanks for the reply. The bisulfite is in a powder form and is prepared fresh each time using milliQ water. I am not convinced that is the problem though b/c even the brand new kit does not work.

The controls from chemicon did not work either, and I assume they should be optimal in terms of how they were isolated since they were from the same company that provided the kit...

Thanks for the suggestions!

Hi Mae,

if this is the case, I would say that the PCR itself has failed, maybe try new batch of Taq and fresh dNTP's and the like.

If your positive controls ain't working, I will have to say it is because of your PCR.

good luck!

Nick

For anyone else who is experiencing CpGenome woes...

I called technical support at Chemicon today, and it turns out that they have been having issues with their bisulfite reagent lately. They are currently working to resolve the issue. Kind of annoying but at least now I know it's not just me!

Thank you Mae for sharing this with us. We just bought 3 CpGenome kits and don't know if they are affected.

I had similar problems until i used a brand new batch of dNTPs, the old dNTPs still worked fine with normal PCR.