The actual bisulfite treatment - protocol and troubleshooting (Nov/18/2004 )
Hi,
I have been reading all the posts and everybody keeps recommending denaturing the DNA before bisulphite treatment and a clean up of the product after the treatment. Is this really necessary? I'm using the methylEasy kit and they don't say anything about it at all. My DNA comes from cell lines and when i used the kit the gene that i use as a positive control gave me the results i want from some cell lines and not others. So, i assumed the ones that didn't give the results i wanted were just not methylated. Is that being too presumptious?
gai
gai,
you would probably find that the MethylEasy kit does have a denaturation step initially with the addition of 3M NaOH and incubation at 37C for 15 mins. I think the second one is incorporated into the resuspension of your pellet and incubation at 72C at the end of the protocol, because it is not overted what is contained in the solutions provided it is hard to say.
as wiith your results, it depends on the gene you have used as a positive control, and you could presume that the methylation status of that particular gene is different depending on the cell line.
I have certainly seen this to be so with the number of genes I am looking at.
Nick
Dear Beverly and all,
I have been working on MSP for two weeks.I followed the protocol which is recommended on this bioforum(Protocol Online Bisulfite Modification (Conversion) of DNA).But It failed include the Sss1 CpG methylase treated DNA.I don`t know what is the problem.My questions are as follows:
1.What is the composition of your Sss1 reaction .My methylase enzyme is purchased from BioLabs.And how to purify thet Sss1 CpG methylase treated DNA.
2.I used a purification kit from TIANGEN company.The 610uL volume is too full to purify because the volume of the column is 800uL.So I used 5M NaCl to bind the DNA and purify it.Can anyone tell me whether it is feasible.
3. The pH of TE buffer I used is 8.5.Does it matter?
4.I am considering Labtechie and your protocol.Can you share me with it?
My email:xuanhq@163.com
Thanks!
Leydig
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 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
hi all,
've been reading through your posts...
having a great time... ...I'm not alone with these methylations and bisulphations...!!
methylnick,
you suggested a methylation-positive control by using methylated plasmids and spiking them in your bisulphite reaction (post#15)... help me,, how can you be absolutely sure that your plasmids will be singlestranded long enough for your bislphite reaction to be completed...
I've been trying to bisulphate plasmids,, unsuccessfully---
plindrm,
to be sure that the plasmids are single stranded there are two things, the NaOH steps in the protocol denatures DNA quite well however for repetitive elements this as well as a higher temperature for denaturation is required. Plasmids should not pose a real problem as it's not as complex as human genomic DNA say.
Also as a sidebar, southern blotting by alkaline transfer (with NaOH) with plasmid spikes as a control lane work very well which suggests addition of NaOH is enough to denature low complexity DNA.
Good luck with it!
nick
am also facing the same problem with bisulphite treatment.... can u tel me the slight modification u hv made in that
The protocol can be found here!
Hi All,
I've been attempting bisulfite analysis for a long time now, starting off with the CpG genome kit from Chemicon in the days before the DNA purification columns, and now using the Zymo EZ DNA Methylation-Gold Kit.
The first results with the Zymo kit are weird. There seems to be only moderate conversion of Cs in all the CpG dinucleotides in commercial unmethylated DNA (combined C/T peak). However, all non-CpG Cs appear as Ts. The methylated DNA makes sense - there is no conversion in the CpGs. The only explanation that makes some sense to me is incomplete denaturation, but I would have thought denaturation at 98C for 10 mins would be enough for any DNA sequence to melt (500ng DNA). Maybe mixing while at that temp would keep the strands apart for the actual bisulfite reaction at 64C. Has anyone had similar problems?
The Chemicon kit worked for my control gene used with the Zymo kit, but with my test gene I wasn't getting reproducible results with directly sequencing the PCR products. Someone has pointed out where I was probably going wrong so I might go back to doing it this way and try again, cloning the PCR product before sequencing. Does anyone know the consensus on how many non-converted Cs are allowed?
Thanks,
Debbie
Has anyone used the Qiagen Epitect Bisulphite kit? If so, any good results? Did anyone have to modify the protocol to get good results? Starting DNA amount? any input would be great.
TPB