Epigenetics

MOD50 Troubleshooting Questions

1. Can REDTaq® ReadyMix™ PCR Reaction Mix (R2523) be used with this kit?
Yes, this kit should work with any polymerase.

2. For the BSA solution in the 2nd step, can a researcher use tRNA or glycogen protein?
Yes, we chose BSA because it was a Sigma product and is less expensive. All carriers we tested, including tRNA and glycogen, performed the same with none showing any advantage, or disadvantage, over the others.

3. What is the best solution in which DNA should be dissolved before using the kit?
You can use 1X TE to reconstitute the DNA.

4. In step 1, should the solution to be used be the one that contains ethanol?
Yes, if this is in reference to step 1 of the clean-up part of the procedure.

5. Does the DNA need to be digested with restriction enzymes before use with this kit?
No, but if restriction enzyme digestion is required, it should be done before bisulfite modification due to sequence changes (C à U) that will occur.

6. Do you need to do anything special for DNA purification?
Before bisulfite modification, DNA should be of high quality. After bisulfite modification just use the reagents supplied in the kit.

7. Can we provide beta actin primers sequences and reaction conditions?
The sequences of the Beta- Actin primer set used for that gel are:

Forward = 5'-GGA GGT AGG GAG TAT ATA GGT-3'
Reverse = 5'-CCA ACA CAC AAT AAC AAA CA-3'

Cycling parameters:
94°C for 2 min. (1x)
94°C for 15 sec.
60°C for 15 sec.   (45x)
72°C for 30 sec.

8. Can the reactions be frozen after the modification and cleaned up later?
Since the DNA is in a fragile state after modification, it is recommended to proceed directly to clean up before storage.

9. How much DNA degradation occurs in bisulfite conversion?
Depending on the quality of the input DNA and which procedure is used (one step vs. two step), degradation can be as low as 40% or as high as 90 - 100%. This modification method is inherently very harsh so degradation will always occur to some extent.

10. Will the MOD50 kit work with RNA samples?
No. MOD50 will not work with RNA. as the modification is done under basic conditions and heating RNA with base will destroy the RNA.