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Help about BSP question. - (Dec/13/2010 )

Hi, everyones,
I start BSP for the methylation status of Promoters. But now, I encounter the some problems as following:
I can not make sure question from conversion process, or PCR process.

I use the traditional method as with 16-18h conversion time

1. Digest 3 µg of genomic DNA
2 Dilute DNA to final concentration 50ul
3. Denature the DNA by adding freshly prepared NaOH (3 M) to a final concentration of 0.3 M. Incubate at 42¡ãC for 30 min.
4. To microcentrifuge tube add:
o 1020 µl 40.5% sodium bisulfite
o 60 µl 10 mM hydroquinone
o 110 µl DNA (+ NaOH)
o 10 µl water

5. Gently mix . Cover the tube with aluminum foil to shield from the light.
6. Incubate at 55¡ãC for 16-18 h.
7. Purify DNA using kit from promega
8. After purification, resuspend DNA and add Water to a final volume of 100 µl.
9. Denature the sample with freshly prepared NaOH (as above) and incubate at 37C for 15 min.
10. Neutralize by adding 10 M ammonium acetate 30ul
11. Precipitate the DNA with three volumes of ethanol, centrifuge for 10 min (14,000 rev/min) at room temperature, wash twice with 70% ethanol and dry under a vacuum. Resuspend in 50 µl water, and store at -20C wrapped in foil.

The primer designed by Methprimer is not working very well. I got some bands but the size of bands (about 220 are smaller the expected size(about 280). After sequencing using PCR product (after extracting gel), it is not my target sequencing

Now I want to use Methyl Primer(ABI) to design. Pls, Give me some suggestions about it. I will appreciate it.

-Yang Qiyuan-

Your protocol looks okay. Multiple bands after pcr could be inefficient pcr primer design or pcr conditions. You would need to be sure your primer design is good and specific and then spend some time optimizing the pcr. Redesigning primers with methylprimer express is a good start. Good luck.

-methylnick-

methylnick on Tue Dec 14 07:19:55 2010 said:


Your protocol looks okay. Multiple bands after pcr could be inefficient pcr primer design or pcr conditions. You would need to be sure your primer design is good and specific and then spend some time optimizing the pcr. Redesigning primers with methylprimer express is a good start. Good luck.

Thanks I will do that, I appreciate your nice answer!!

-Yang Qiyuan-