Hello everybody,
I am performing Paired End BISULFITE sequencing, with a modified Illumina protocol. I do the Covaris shearing, end repair, adenylation, ligation and size selection (E-gel), followed by bisulfite treatment using the EZ DNA methylation kit.
I then performed the elution in 30 ul EB solution (according to the protocol) but I recently ran into some problems in the next step (PCR).
I use 1-3 ul of bisulfite-treated DNA for PCR and, even if it looks I have enough DNA for seq, the library complexity is not enough to guarantee good results.
I then thought that, if I use more DNA for the PCR I will get more but, on the contrary, I get even less!
It seems there is an inverse correlation between input DNA and amount of PCR product. Could it be due to an inhibiting effect of the EB solution? Should I rather do the elution in TE or water?
Finally, it is well known that sodium bisulfite damages the DNA but I also noticed that it seems there is no relation between amount of DNA after bisulfite treatment and after PCR, as if not all the DNA is "amplifiable". This makes it very hard to predict the amount of input DNA and number of cycles needed for PCR. Does anybody have seen the same thing?
Thank you very much!
I am performing Paired End BISULFITE sequencing, with a modified Illumina protocol. I do the Covaris shearing, end repair, adenylation, ligation and size selection (E-gel), followed by bisulfite treatment using the EZ DNA methylation kit.
I then performed the elution in 30 ul EB solution (according to the protocol) but I recently ran into some problems in the next step (PCR).
I use 1-3 ul of bisulfite-treated DNA for PCR and, even if it looks I have enough DNA for seq, the library complexity is not enough to guarantee good results.
I then thought that, if I use more DNA for the PCR I will get more but, on the contrary, I get even less!
It seems there is an inverse correlation between input DNA and amount of PCR product. Could it be due to an inhibiting effect of the EB solution? Should I rather do the elution in TE or water?
Finally, it is well known that sodium bisulfite damages the DNA but I also noticed that it seems there is no relation between amount of DNA after bisulfite treatment and after PCR, as if not all the DNA is "amplifiable". This makes it very hard to predict the amount of input DNA and number of cycles needed for PCR. Does anybody have seen the same thing?
Thank you very much!
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