The actual bisulfite treatment
Posted 18 November 2004 - 11:45 AM
Does anyone have a "favorite" protocol they'd like to share? My lab has suggested always using TE instead of water (to protect the DNA) and to find out better ways of cleaning up the DNA from the column. I am not in favor of a kit as I see no reason to use "reagent 1" that is really NaOH and "reagent 2" that is just bisulfite at a 400% markup.
Any advice you can offer would be MUCH appreciated. I have been spinning my wheels for four months now and I only have this job for a year! I need results!
Posted 18 November 2004 - 03:34 PM
First, loss of DNA during purification is one of the many possibilities for failed PCR, especially with Promega Wizard kit. I think using some bisulfite modification kit will help such as the kit from chemicon because of the built-in purification method.
Second, cycle your PCR up to 40 times if you have not done so.
Posted 06 January 2005 - 11:24 AM
Posted 06 January 2005 - 05:48 PM
Posted 07 January 2005 - 06:19 AM
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.
Posted 08 January 2005 - 09:13 PM
If you have got good PCR products, cloning and sequencing is not a big deal. Just purify your PCR products using Qiagen PCR or Gel purification kit and do a 5-min TA cloning. Pick 10 clones for sequencing (I have found ten represents well the actual profile and no more is needed).
Posted 11 January 2005 - 09:00 AM
Thank you SO much for all your help. I think I'm confused as to how I get from the ten clones I'll pick to the sequence-- is it a miniprep followed by dideoxy sequencing? Also, how do I get the clone off the plate? Any advice would be much appreciated, as my boss is out of town and I have no idea what I am doing.
Edited by labtechie, 11 January 2005 - 09:01 AM.
Posted 11 January 2005 - 10:46 AM
Hope that helps.
Posted 25 January 2005 - 02:41 PM
labtechie, on Jan 7 2005, 07:19 AM, said:
I am not exactly sure about the chemistry of the bisulfite reaction and if NaOH is enough to desulfonate the adducts.
From my understanding, NaOH helps denature the DNA strands to expose the sulfonated adducts to another chemical such as ammonium acetate.
That could be a reason why you are not yielding converted DNA.
Please correct me if my understanding of the reaction is wrong anyone!
Posted 26 January 2005 - 05:54 AM
Posted 26 January 2005 - 02:18 PM
labtechie, on Jan 26 2005, 06:54 AM, said:
Posted 23 February 2005 - 06:15 AM
Posted 23 February 2005 - 10:10 AM
Just curious about all these things... so glad your protocol works!
P.S. All: I do NOT pre-digest my DNA with a restriction enzyme and this protocol still works FINE.
Posted 23 February 2005 - 02:46 PM
labtechie, on Feb 23 2005, 11:10 AM, said:
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