Over the past two/three weeks I have been busy with the bisulfite conversion. I used a Qiagen kit and its protocol to perform the procedure. However, although the protocol is straight forward, I have yet to have any success. My first conversion consisted of extracting the DNA, converting it using the kit, and amplifying the DNA using PCR. However, throughout one of these processes something went wrong. So.... Bryan and I started troubleshooting:
-The PCR Cycles- we tried different time cycles to see if that has an effect on the primer binding to the DNA
-The original Genomic DNA- we ran this through an agarose gel to make sure we had DNA to begin with
-The annealing Temps.- we ran the bisulfite converted DNA through a PCR machine with a gradient. We could then see the optimal annealing temperature.
-My technique- we will soon do the procedure side by side.
Despite testing for all of these issues, every time we ran the converted/amplified DNA through a gel we did not see anything in the gel, except for Primer Dimer, which tells us the the primer is not binding to the DNA. I attempted this conversion six times and ended with no results. I was frustrated, but Bryan told me, "thats Science- lots of Troubleshooting." Thankfully, I have moved on from that conversion. Eventually Bryan and I will do it side by side, to test for any human error.
Now I am doing a western blot and will update the blog as soon as I get the results.
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