I already optimized methylation-specific PCR (MSP) and got results for 50 samples. However when I repeat it again using a new bisulfite treatment kit, I cannot get consistent band.
Below are the detail situation when I use the new bisulfite treatment kit.
When I did for first time the bands appear very clear and thick. But when I repeated 4 times treatment and about 10-12 MSP, mostly no bands appear. If they appear they were faint and smear. I tried to change the MSP reagents (AmpliTaq gold polymerase, buffer, MgCl2, dNTP, primers) to the latest one I got less than 6 months ago but still don't get any result.
The concentration of treated DNAs were above 12 and purity mostly 2.1-2.7. Only one time purity was 0.17. Initial DNAs for treatment are of excellent concentration and purity.
I really appreciate if anyone can suggest any idea to get the band. I just want to amplify 4 samples for DNA sequencing.. already took few months to do..
Your reagents need to be made fresh just prior to use.
3 M NaOH
0.12 grams of NaOH
1.0 ml of autoclaved dH20
10M NaOH
0.40 grams of NaOH
1.0 ml of autoclaved dH20
40 mM Hydroquinone
0.55 grams of Hydroquinone
12.5 mls of autoclaved dH20
Bisulfite Solution
8.1 grams of Sodium Bisulfite
15 mls of autoclaved dH20
add 1.0 ml of 40 mM Hydroquinone
pH to 5.0 using 10 M NaOH (150 µL)
Hi,
If you are manually converting your DNA, the suggestion of Timothy is correct, since the bisulfite conversion will be highly impaired by old reagents.
Also for manual conversion make sure you clean your DNA thoroughly since the the high salt concentration will impair your PCR.
If you are using a commercial kit, the issue should be in your amplification step or in the way you store your bisulfite treated DNA.
Bisulfite DNA needs to be stored at -20°C for medium to long term storage (some people even suggest -80°C for long term).
+4°C is ok for several hours but I would not use this for longer period.
At room temperature your bisulfite DNA will degrade really rapidly.
For your amplification, not all the taq will give you good results. If you can afford it, you might want to try another taq that the amplitaq gold. I used it in the past and also got an issue or reproducibility and specificity with bisulfite treated DNA.
After changing to platinum Taq, I got stronger and specific bands. Amplitaq gold is a good product for regular DNA, but I expect it does not like too much the low complexity sequences which result from bisulfite conversion.
Be aware that some taq does not use template with uracyles... the high fidelity Phusion Taq for example does not work on Bs-DNA. Also last point, if you change brand of taq make sure your taq does not contain UNG...
I hope this is useful,
Best,
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