The actual bisulfite treatment - protocol and troubleshooting (Nov/18/2004 )
Hola!
I've tried this agarose-beads-based protocol for the bisulphite treatment, and I've got a little question about that: How do you prevent the beads from melting during incubation at 50°C? I get beautyful beads in the mineral oil, however, whenever I go to 50°, they disappear... I must admit that I didn't adjust the pH of the bisulphite-hydroquinnone-solution, as I'm just beginning with dry practices without DNA. My logic tells that that's not the point, as the LMP-agarose has a melting point < 40°C and will thus melt anyway.
Would be so nice if you would have an answer to this!
Greetings
Biotom
1. Set 300 ul mineral oil (I use Sigma M5904) in 2 ml tubes on ice for 30 minutes.
2. Prepare less than 1ug DNA (you may digest it with a restriction enzyme that has no site within your region of interest) in a volume of 21 ul TE.
3. Add 4 ul of 2M freshly prepared NaOH and incubate 15 minutes in a 50C water bath
4. Make a 2% LMP agarose solution (seaplaque agarose from cambrex) and add 50 ul of it to the 25 ul of DNA solution
5. Before it cools, form beads by gently pipetting 10 ul into the middle of the mineral oil layer (only one bead per tube—therefore up to six tubes per sample)
6. Leave for at least 30 minutes
7. Prepare bisulfite solution (avoid light by wrapping tube in foil): 3.8 g sodium betabisulfite in 5 ml H20 (this is 5 M) and also prepare 110 mg hydroquinone in 1 ml H20. Mix these two solutions, check pH, and adjust pH to 5 with 2 M NaOH.
8. Add 500 ul of this solution to tubes—this is the important part. Pipet very gently to get the layers to separate correctly and check that your bead is in the aqueous layer. Other protocols say “invert”—that doesn’t work.
9. Cover tubes with foil (light exclusion) and incubate in hybaid for 4 hours at 50C.
10. The following steps require both p1000 and p200 pipets (avoid coming near the bead with the tip of the p1000, it may break it): remove oil and solution and rinse with 1 ml TE ph 8, 4 x 15 minutes (I use a rotating tray).
11. Desulfonate with 2 x 15 minutes in 500 ul 0.2 M NaOH.
12. Stop treatment in 1 ml TE 2 x 10 minutes, store overnight (or a little longer) in 1 ml TE.
13. Before PCR, rinse with 1 ml H20, 2 x 15 minutes. Use one bead (something less than 100 ng) per PCR reaction
14. PCR hints: Use a hotstart taq with NO PROOFREADING ABILITY—I use Faststart from Roche, it’s cheap. Also do a nested PCR and do not expecct any products from your large primers.
I am now working on cloning and sequencing these products, so if anyone has any hints.... they'd be much appreciated. I'm using the PCR-script cloning vector from Stratagene.
-labtechie
Biotom,
I have to admit I don't use low melt agarose in my beads. I never saw the point to purchase a different kind than the one I used for other applications. I use a hot start PCR enzyme so the bead melts no matter what when the PCR begins. As far as I can tell, the low melt doesn't confer much, if anything, in the way of advantage over regular agarose.
I can't really come up with anything helpful to say about the melting other than switch agarose. Unless... When I do the incubation, I did notice that the beads got more clear than they were before. Are you checking for the beads visually or are you using something to stick into the tube and see if they are there? The are soft at that temp no matter what. Once you put the first wash on the bead they harden back up and are a little bit cloudy again.
Hope that helps,
Beverly
Dear All,
I used the Modified Olek Protocol (by Labtechie-Thanks for it.) for bisulphite Modification of DNA isolated from white blood cells. I use 4ul (of 300ng/ul) of DNA. The Beads look fine even after TE washes, But I got no PCR products 
with primers for the modified sequence for my gene of interest.
My Primers are 25 bases long designed with meth primer (with no CpG's) and I use a Promega GoTaq Green Master Mix.
I saw that it you have all advised Nested PCR, and a Hotstart PCR mix, but does anyone get products without a nested PCR? I mean do you guys see products after the first round of amplification?and with any other other Master-Mix?
Should I make the primers shorter and try using to 15 mer primers with the second internal to the first (Nested).
Would buying the Faststart Mix from Roche help?
I would be grateful for any advice, as it ism an undergraduate project and I have less time to finish it !!!!!
Thanks a lot,
Methstarter
Hello,
I used to run my first round PCR on the gel too, just to see what was there. There was never anything I could see. I think you end up with so few copies on the first round, that is why everyone does nested PCR. I started using nested PCR because I had three seperate promoter sections I was looking at. I would do a first round that encompassed all the sections, then three second round PCRs that would each have that first large product as template. Seemed to work great even though I never saw any product on the gel after the first round.
I recommend nested PCR.
Beverly
Hi,
I am a new in the field of methylation analysis. I tried the posted protocol, cloned and sequenced PCR products: I did not get any C to T conversion whatsoever. Something must have gone wrong with the chemical reaction. Does anyone have an idea what the reason might be?
By the way: one protocol describes that 30 microliter 10 mM hydroquinone should be added to a total volume of 610 microliter (makes it less than 0.5mM), while in the agarose-bead protocol 110 mg hydroquinone are in a final volume of 6 ml (makes it 167 mM). Quite a difference; can anyone explain this?
Thank you
Julius11
julius, there could be a number of reasons why you did not get C to T converison and may not have anything to do with your conversion. But to eliminate this, did you denature your DNA properly prior to conversion...as this is essential to ensure that the bases are exposed to the bisulfite for the reaction to occur.
If this has been ensured, then the next step, primer design for PCR amplification is essential, you must design primers in a way that favors the amplification of converted templates, and this is discussed else where,
As for hydroquinone, I have found that the amount of this does no drastically effect the outcomes of the reaction.....it turns out that hydro quinone is used in photography and developing film and my take on it is that it is used to stabilse the bisulfite in solution during the reaction......i could be wrong, I normalling you an excess of hydroquinone and my reactions have been working fine.
Nick
Hi, labtechie:
I use your protocol ( agrose) to do bisulphate PCR. (nest PCR )
But I found that most sample work well, but 20-30% samples donot work. I donot know why.
Now, I add 20 water into one agrose bead, 80 degree to resuspend it for 2 min, then add 3 ul modifed DNA to 20 ul PCR system. And second time, I use 1 ul first PCR product.
Can you give me some advice about how to improve it?
Thanks
Do most people reading this buy fully methylated DNA or is there a region I can amplify that is known to always be methylated? Also, the only artifacts I get are sometimes a non-converted nonCpG C a few bp away from a CpG. Do you get that? Interesting artifact, a result of steric interactions or secondary DNA structure maybe?Hi Ellen,
as a positive negative control, instead of buying methylated DNA it might be worth your while to methylate some DNA yourself. You can use SssI methylase (NEB #M0226) and this enzyme methylates every CG residue available. As a control I would methylate a plasmid such as pGEM and then "spike" my sample with the methylated plasmid prior to bisulfite treatment. You can assume that all CG's on the plasmid are methylated after SssI methylase and therefore should all come up as CG after bisulfite sequencing.
As for non-CG non conversion close to CG residues, this is due to incomplete denaturation of the strands and thus inefficent bisulfite conversion.
Hope this helps
Nick
Hello labtechie
I have some question about the protocol. What is the chemical reagent "sodium betabisulfite" ?
I can't find it from biology company. Is it sodium metabisulfite?
Another question, can I use water instead of TE? Why do you use TE but not water?
The third question is that you keep the bead in TE overnight at 4C? And after that do you keep it at -20C?
I need your help. Thank you!
xiaohai
Using Microcons is fast and allows many samples to be treated in parallel:
http://methdb.igh.cnrs.fr/cgrunau/methods/...te_engl_mc.html
Good luck.
any benefits to doing the bead method over a kit? Zymo kit is working great for me, but if this is more efficient in some way that would be something.
I kinda want to try it, but i need a reason. hehe...