First week back in the CS lab!
I actually started back in the lab on May 29th and, go figure, Baby Huddleston decided he was ready to meet his family! Of course, Dr. Huddleston, who was in labor, was worried about running a gel on a vector she made and asked me to do it for her. That lady. I was more than happy to do it for her! It felt so good to be back in the lab! I really missed it in there. It was so nice because nobody else was in the lab so I shut the doors and played my music loud. I started initial PCRs for my SOE PCR that week. I got 2 out of my 3 of my needed gene combo products done!
So, the official first week back in the lab and there’s a lot of new changes! First, meeting at 8 am (really bad since I’d been waking up at 4pm every other day). Second, I had two new lab partners (since Kristin and Zach left me to be adults and go to dental and medical school. Whatever.). Third, Dr. H was out being a good mom. And fourth, I was bestowed the honor of showing the new kids the way around the lab. They’re Madeline, my dear friend who lived across the hall from me freshman year, and Jacob, a freshman sophomore who is actually the sweetest. I am so excited to spend the summer with them.
We’re beginning the summer with SOE (splice overlap extension) PCR . I am so excited because we’ve been talking about doing SOE for over a year and we’re finally here! On our agenda this week we needed to do the initial PCR of their products so we can sew (get it??) them together. We’re replacing our genes with a gentamicin cassette to be used for screening. It’s sort of bizarre teaching people how to get around the lab because it’s second nature to me now. They were asking me how to pipette product into the wells in a gel, and I stood there for a minute just going… uhhhhh. I had to watch myself do my technique so I could articulate it to them. Funny how far I’ve come. I was in their shoes a year ago. Poor guys are stuck with me and not Dr. H!
Once we obtained and confirmed our initial products, they were purified to be used for the second step of SOE PCR: the first SOE reaction. This will bind initial products A and B to each other. Both Madeline and I encountered the same problem: we obtained products AB of the correct size, 1kb, but we also had products that were 500bp, which indicated to me that the pieces of DNA were breaking up into their initial products, A and B, or they weren’t binding together at all and the gel was picking up the original amount of template added to the PCR reaction (thanks Dr. Brokaw).
So, the plan was to change the annealing temperatures and extension times to see if we obtained better results for the next week. Until…