A new protocol:High-speed conversion of cytosine to uracil in bisulfite genomic - (Oct/12/2006 )
Bisulfite Modification (Conversion) of DNA
By tao
11 October 2006
1. DNA extraction using phenol/chloroform/isoamyl alcohol and proteinase k.
2. Dilute 1 µg DNA (no more than 2 µg) in 18 µl TE buffer(10mMTris•HCl, pH7.5/0.1mM
EDTA,pH8.0).
3. Pass the DNA through a narrow-gauge needle several times to shear the DNA.
4. Denature the DNA by adding 2 µl freshly prepared NaOH (3 M, final concentration 0.3 M).
Incubate at 37-42°C for 15-30 min.
5. The preparation of 10 M bisulfite solution : Briefly, 2.08 g NaHSO3, 0.67 g (NH4)
2SO3•H2O, and 5.0 ml 50% (NH4)HSO3 were mixed and heated at 90°C to obtain a
solution of pH 5.2–5.3 (measured at ambient temperature).
6. Treatment of DNA using 10 M bisulfite solution was performed as follows: 218 µl of 10 M
bisulfite solution (maintained at 70°C) or 226 µl (maintained at 90°C) was added to the
alkali-denatured DNA solution (20 µl) .Each mixture was incubated for a designated
period in a water bath maintained at either 70°C or 90°C (mineral oil-overlaying was
not performed, nor was hydroquinone addition). Gently mix. Cover the tube with
aluminum foil to shield from the light.
7. Purify DNA using the Promega Wizard DNA purification kit.
8. After purification, resuspend DNA and add TE to a final volume of 50 µl.
9. Denature the sample with freshly prepared NaOH 5.625 µl (as above) and incubate at
37°C for 15 min.
10. Neutralize by adding 10 M ammonium acetate 23.9µl (pH 7.0) to a final concentration 3
M.
11. Precipitate the DNA with 160 µl (two volumes) 96% ethanol, cool DNA (overnight, -20 °
C) ,centrifuge for 10 min (12,000 RPM) at room temperature, wash twice with 160 µl
70% ethanol and dry. Resuspend in 20 µl TE, and store at -20°C wrapped in foil. The
treated DNA should be used within one month as degradation may occur in the cleaned
and frozen sample.
12. The resulting DNA now can be used for PCR such as bisulfite genomic sequencing PCR
(BSP), methylation specific PCR (MSP).
This protocol is the newest .I havn't seen anyone use it except the author of original paper.
The original paper :http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed
or find the paper attached
Please check my protocol seriously and try to find some problems! I will appreciate everybody's advice and assistance!
Hi Tao,
what problems are you having exactly?
it's an interesting protocol that can be performed within 40 minutes.
Hi methylnick,
Thanks for your immediate reply and concern!
Yes,it is an interesting protocol.As far as I know,It seems nobody knows this new conversion method.I try to use this quicker method to research on DNA methylation.But I can't get any PCR products until now.I don't know whether there is problems in the conversion protocol or in the subsequent PCR conditions. I refer to primers and PCR conditions in the original paper.So I think my conversion protocol is problematic.I need your help.Could you check my protocol seriously and try to find some problems!
many thanks!
I haven't tried this method myself, but with the conventional method, a carrier is added for DNA precipitation, you could add 1-2ug of glycogen to your DNA solution for precipitation and this would enhance your DNA yield after bisulfite.
good luck
Nick
Thank you! Nick,I will try your advice.
Other questions:
1.Is restriction enzyme essential?using of small-gauge needle can do the same function?
2.What is the difference between tRNA and glycogen?
3.How can I check the quantity and quality of the end yield conversion DNA?
Could you give me your e-mail or QQ?So I can consult with you directly.My e-mail address is huitaofan@yahoo.com.cn.
Thanks a lot!
Either or is fine, it's best to reduce the complexity of your starting gDNA. I use a 19G syringe needle as restriction enzymes may limit the number of loci you can assay for.
Their utility is about the same, to help "precipitate" DNA. Again either or will work
This is a difficult one, the only way is to perform a bisulfite PCR. To measure conversion, or in this case amplificiation efficency, a restriction digest is normally performed and you are looking for either the loss or the gain of a restriction site after bisulfite conversion.
Good luck!
Nick
ps: I have pm'ed my email address