The actual bisulfite treatment - protocol and troubleshooting (Nov/18/2004 )
Thanks! That makes me so happy, that my protocol is working so well for people. Just wanted to tell you all that I no longer work in that lab, so I'm not so up to date with questions people might have. I have NO idea how you'd reference my protocol, especially since I did not get publishable results from my methylation experiment and thus have no publication with my modified protocol in it. It would be nice to get credit. Anyone have any ideas?
Ellen
EllenHow about:
Labtechie (your real name), Personal communication.
or
Labtechie. The actual bisulfite treatment. BioForum http://www.protocol-online.org/forums/index.php?pid=23401
Lastly, thank you labtechie for your contribution to the community.
Good luck with your future career!
Dear Labtechie and all,
Thanks for the protocol. I tried your method and it works. The Chemicon kit didn't though. Hmm, I wonder why.
Anyways... I have a question. I use 1 bead per 20ul PCR reaction, as suggested. After the PCR, I find though, that the reaction solidifies really quickly (since it's a small volume and it's 2% gel). How do you load your PCR reactions into electrophoresis wells? Just like really quickly? Or is there a solution I can add to the mix at the end of the PCR to keep the agarose in molten state at room temperature?
Many thanks!
I am using the Chemicon Fast kit and it seems work very well and very easy to use.
Hi All,
I'm a starter with methylation work only 2 weeks old. I am working with nanogram quantities of human DNA obtained from cheek cells. After the bisulphite treatment , the DNa is complexed with a lot of sodium so i'm thinking of buying a promega wizard DNA clean up kit which i read that the pcrman and many of u use, Do you think I should use that to purify, or buy a kit which does both bisulphite and purification. As I plan to use COBRA (Combined Bisulphite Restriction Anlysis) for finally anlysing my region of interest. Sorry about my questions being dumb, just too new to the whole thing.
Cheers!
methstarter
Hi methstarter,
I would suggest that you buy a kit which does the modification and purification. The promega wizard DNA clean up kit is not good in terms of DNA recovery.
Good luck!
methstarter,
you starting quantities of genomic DNA is very low for successful bisulfite treatment via conventional means. I would suggest a dedicated bisulfite kit that does not degrade DNA in the process. One I have used with great success is methyleasy from Human Genetic Signatures I am aware Chemicon also sell a bisulfite kit also.
As pcrman eludes to, the wizard kit is not good in terms of DNA recovery and the conventional method of bisulfite treatment can degrade upto 90% of your starting DNA!!!
good luck!
Nikc
Dear pcrman and methylnick
Thanks for your advice. Its great to have ppl like u to ask.
I think I will buy one of methyleasy or chemicon, whichever my supervisor suggests.Will get back to you.
Cheers,
methstarter
Thanks for the protocol. I tried your method and it works. The Chemicon kit didn't though. Hmm, I wonder why.
Anyways... I have a question. I use 1 bead per 20ul PCR reaction, as suggested. After the PCR, I find though, that the reaction solidifies really quickly (since it's a small volume and it's 2% gel). How do you load your PCR reactions into electrophoresis wells? Just like really quickly? Or is there a solution I can add to the mix at the end of the PCR to keep the agarose in molten state at room temperature?
Many thanks!
I also have used a modification of the Olek protocol. Slightly different than the one from labtechie. I would form my beads in a 12 well plate, one well for each type of bead. Then fish them out with a little scooper and put them in microfuge tubes. I would then leave them in the fridge until I needed to use them (no problems for quite a while even with no covering for the bead). Then I would treat in sodium metabisulfite and hydroquinone (that other person is quite right about the solubility thing, I always made a lot of hydroquinone and only added a little bit to my reaction mixture). Then I would treat the beads overnight at like 50 C. The washes were relatively the same. As with desulphonating. Sometimes I would wash the last wash in TE with 10fold less EDTA, but I never saw that it made that much difference.
Finally the reason I quoted that thing: The PCR, I would do a nested PCR as I was looking for several sites near each other. I had a long product which I would use as template for three new PCRs with smaller products. So, if I didn't get to the PCR in time and it had hardened before I got the second round started up, I would just mix and poke with the pipette tip until enough came up the tip that I was satisfied. I would run the second round and that would be that. Although if you are just looking for something diagnostic, you can always grab a bit of the hardened PCR and just jam it down in the well in your gel. You don't need to add dye or anything to weight it down, just stick it in there and run your gel. The DNA will run right out of your hardened PCR into the gel. I have done just that thing before. Also I have stuck whole beads into wells on a gel. That works too.
If anyone is interested on the exact details in my modification of Olek's protocol, I can go and find them.
Beverly
Dear Beverly,
Thanks for the tip!
So far, after my PCR, I gel extract my products out from the hardened mix. But you are right, just sticking the hardened PCR directly into the well should work; saves reagent, steps and time!
Labchick.