Troubleshooting

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.

Lab Techniques

Over the past three weeks Brian has been showing/teaching me the lab techniques I will need to know for my project. My first few attempts, even with the guidance of Brian, were somewhat failures. Aside from learning the steps, my data did not match what was expected. The very first experiment I ran was extracting the RNA. RNA is extremely fragile and not as stable as double stranded DNA. Because of this I had to use (RNA-Free) lab tools. My pipettes needed to be clean and safe for the RNA to remain intact. However over the course of extracting the RNA, running it in a PCR and loading it into an agarose gel, I must have messed something up, because when we went to take a picture of the gel, there was no result at all. Brian admitted that for my first time doing a PCR, the amount of samples may have been too much. So, the next day, I ran another one with only a few samples, and finally we saw some results-results that Brian had already gotten in his previous lab work. Science can be very tedious. One mistake can set you back to the beginning of the experiment. Regardless, I have been learning a lot. I have several techniques down: RNA extraction, PCR with RNA and DNA, electrophoresis with 1.5%agarose gel, Western Blotting, transferring a western blots to a membrane, and developing the membrane in a dark room. Just recently I have been working on a bisulfite conversion. Over the last few weeks, Brian has taken a few steps back and now allows me to complete the work with little assistance. I do not have to ask him as many questions, and he is beginning to trust that I am following the protocols correctly. Overall it has been an exciting/edifying experience, and I look forward to learning much more as the days continue.

I will upload some pictures of the lab and some of my results with explanations soon.