Bisulfite DNA modification protocol - (Sep/02/2005 )
This protocol was originally posted by Methylnick as a Word file. The original post can be found here http://www.protocol-online.org/forums/inde...?showtopic=9241
Hi Nick,
Thank you for sharing your protocol with us. I have converted it into html file and post it below so that it is easier for others to view it.
Bisulfite Modification Protocol (by Methylnick)
1. Digest target DNA with restriction enzyme that does not cut the fragment of interest. (Post clean up and isolation). Phenol/chloroform extract and resuspend in water. Or, shear DNA by passing through a narrow gauge needle (21G) in lysis buffer. Shear DNA in a total volume of 150 µl with ten strokes. Take approximately 2 µg of DNA from this solution for bisulphite treatment.
2. Denature DNA (2 µg) in a volume of 20 µl by adding freshly prepared NaOH (3 M stock, final conc. 0.3 M) incubate 15 minutes 37°C. (make DNA solution up to 18 µl then add 2 µl 3 M NaOH)
3. Prepare fresh solutions of 10 mM hydroquinone (quinol is equivalent and was used here). 0.55 g into 50 ml of water (water for irrigation, sterile) to make a 100 mM stock solution, then dilute this down to 10 mM. It was discussed at “lab chat” that if you use the quinol solution neat (100mM) there were no adverse effects on the bisulphite reaction. Quinol is used to prevent the oxidation step of the reaction (check). Make 3.6 M solution of sodium bisulphite (sigma) or 2 M sodium metabisulphite (BDH), pH 5.0 (pH adjustment with 10 M NaOH). BDH metabisulphite was used. Made a "20 ml" solution of bisulphite by taking 7.6 g of sodium metabisulphite added 18 ml of water (for irrigation) and then pH the solution by adding 416 µl 10 M NaOH. At “lab chat” it was discussed that you can use a super-saturated solution of bisulphite. You add 15ml of water and pH with 416 µl of NaOH and use that. NaOH helps the bisulphite go into solution, but with a super-saturated solution, it is okay to have a few granules at the bottom of the tube. There was also discussion about the amount of NaOH to use for pH. 208 µl was a figure thrown around. Jenny and I used 416 µl and checked the pH with their new pH meter. The reading was 4.97. The margin of error from the calibration solutions (and maybe the meter itself) is ±0.02 so it is within a pH of 5.0. Use a final concentration of the bisulphite ion to be 3.1 M.
4. Add 208 µl of sodium bisulphite (Sigma) or sodium metabisulphite (BDH), 12 µl of hydroquinone to a final concentration of 0.5 mM to the denatured solution of DNA. Final volume is 240 µl.
5. The reaction was vortexed lightly and overlayed with mineral oil (sigma equivalently used for PCR) and incubated at 55°C in the dark for 16 hours. The reaction was set up by 6pm and finally extracted at 10am the following morning.
6. Recover bisulphite DNA from under the oil. This was done by pipette. The reaction mixture was extracted from beneath the oil. Wiping the tip with a tissue to remove the oil. Some of the reaction mixture was left behind (around 5 µl). What was said in the original protocol is to snap freeze the reaction and remove the oil (I have tried this in the past and it also works).
7. The free bisulphite was desalted using Promega Wizard DNA Clean-Up System. The resin was stored in the absence of light (the PCR resin is light sensitive so the DNA resin was also kept in the dark). The DNA was eluted with 50 µl of water (for irrigation) that was heated to 70°C.
8. Denature the DNA in solution by adding 5 µl of 3 M NaOH to give final concentration of 0.3 M NaOH. Sample was incubated for 15 minutes.
9. Neutralise solution by addition of 5 M NH4Oac, pH 7.0 (ammonium acetate) to a final volume of 3 M. This equates to 33 µl. The ammonium acetate acts to desulphonate the uracil derivative to uracil for PCR. The sulphonated uracil derivative is inhibitory to PCR.
10 The DNA is precipitated from the solution by the addition of ethanol. (2 x vol). The tubes were then incubated on dry ice for >20 minutes. In our case it was over 2 hours (lunch!). The tubes were then centrifuged at maximum speed for 20 minutes to pellet DNA. The supernatant was decanted out. The tubes were centrifuged again maximum speed for 5 minutes; the residual supernatant was pipetted out of the tube. The pellets were left to air dry (we did not use a wash step) the pellets were visible.
11. Pellet was resuspended in 50 µl water and can be stored at -20°C. The tubes were left at 4°C overnight as a way to resuspend the DNA into solution. The original protocol suggests 0.1x TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) and storage at -20°C. There is no need for yeast tRNA although it has been used in the past. With our case it has not been used.
Recipes:
2 M Sodium Metabisulphite “20 ml”.
7.6 g of sodium metabisulphite (BDH)
15-18 ml of water
pH to 5 with 416 µl 10M NaOH.
Can make to 20 ml but supersaturated solution is also fine.
10 mM Hydroquinone.
0.55 g quinol (BDH)
50 ml of water to make a 100 mM stock solution
Dilute stock down to 10 mM. 1:9
10 M Sodium Hydroxide
4 g NaOH in 10 ml. Make 3 M NaOH by diluting 10 M stock.
cool, thanks for that pcrman 
thanks for that! its been a great help!
cool , many thanks
thanks for that! its been a great help!
cool , many thanks
Hi, I have a small question about the protocol.
In step 9, why the addition of 33 µl of 5M ammonium acetate will result in the 3M final concentration ?
It should be less than 2M, isn't it?
Or I have something wrong calculating it.
Can anyone tell me?
Thanks.
MY
Hi, buddy.I also have the same question about the protocol as monyen.
In step 9, why the addition of 33 µl of 5M ammonium acetate will result in the 3M final concentration ?
It should be less than 2M like that.
I have calculate the quantity of ammonium acetate to make 3M the final concentration.It should be 82.5uL.
Can anyone tell me which one is right?
Thanks a lot!
hi leydig and moneym,
good spotting it's a typo...i have been using 33uL of ammonium acetate and this works well.
Nick