Doug Reinisch Weeks 3 and 4

This is my first blog post in two weeks as both weeks have been shortened by the fourth of July and my grad student Joe’s mini vacation to New York City.  That said despite spending less time in the lab than previous weeks, I still managed to accomplish a great deal of work.

I only worked 3 out of 5 business days during the week of the fourth of July.  After celebrating our nation’s independence over the four day weekend, I went back to work on Wednesday, picking back up where we (Kennadi and I) left off.  Using a QIAprep Spin Miniprep Kit, we isolated the DNA from the bacteria cultures that we grew in LB broth the previous week.  Additionally, this day marked the end of Khin’s (a graduate student) time in the Bouchard Laboratory.  We celebrated her many hardworking years in the lab as we welcomed a new transfer student who will be spending the next 10 weeks in the lab.  It seems like every week someone is either leaving or joining the lab giving me the opportunity to meet so many new scientists from around the world.

QIAprep Spin Miniprep Kit

On Thursday we began by using the nanodrop machine to determine both the concentration and purity of our isolated DNA.  After the nanodrop showed promising results, we cut the DNA plasmids into three subsections (plasmid backbone, Green-Fluorescent Protein (GFP), and the HBx mutant) using restriction enzymes.  FroOn Friday we ran this DNA on a DNA gel to confirm that we had created successful mutant genomes of HBx.  Our success rate was quite high (6/7 samples worked) leaving only HBx 1-98 to be redone.  Joe advised us to pick and grow 6 different bacteria cultures from our agar plates made the previous week in hopes that one or more of these colonies picked up the mutant plasmid.

As shown above, all samples (right of empty well) had at least 1/2 plasmids with a mutant HBx genome (less base pairs than the normal HBx protein in well 2 at the bottom) except for HBx 1-98.

When I came back to the lab on Monday morning we hit the ground running with where we left off on HBx 1-98.  We repeated the same steps that we had done just days before (QIAprep Spin Miniprep Kit followed by a restriction digest on the isolated plasmids).  In the afternoon Nick, a Ph.D. candidate in hopes of graduating by early August gave a presentation at the lab meeting held nearly every Monday.  He discussed his most recent project as Dr. Bouchard and Dr. Stills questioned him on his methods, findings, and future applications.

Nanodrop results for 5 of our 6 repeated 1-98x samples.

On Tuesday July 11, Kennadi and I finished our work with HBx 1-98 as we found that 2 of our 6 colonies (using gel electrophoresis) had been transformed with the mutant plasmid in it. Now that we had successful mutants for all 7 of our original primers, we were ready to send our off for sequencing.  Later that day Joe taught Kennadi and I how to split a collagen plate full of HepG2 cells into two separate collagen plates, creating more room for the cancerous cells to grow and creating a larger sample size to work with.  This would be a process that Kennadi and I would have to repeat on our own on Friday.

Cell plates after being split in the 37 degrees Celsius incubator.

The following day Kennadi and I were on our own as Joe took a 3 day vacation to New York City.  We began by starting a new mutagenesis project (PCR) on a new primer for HBx that Joe had designed.  We had to wait a couple hours until the thermal cycler was done at which point we ran both samples that we created (one by Kennadi and one by me) on a DNA gel.  Unfortunately our gel did not show that we had any genetic material at all.  Anyway Joe gave both of us off on Thursday while he was gone to work on our joint presentation that we will be giving to the entire lab in the coming weeks.

As seen here no genetic material showed up on the DNA gel under the UV light (only the ladder appeared on the left).

The final day of my fourth week in the lab, Joe again was not present, but left us with a list to do on our own.  Kennadi too was missing for the morning part of the work day as she had a meeting with her program’s director.  This left me alone to redo the PCR for our new HBx primers in hopes of having greater success than we had earlier in the week.  After this I waited up for Kennadi to split the HepG2 cells that we had growing in the incubator from when Joe taught us on Tuesday.  Since it was Friday and I got out of the lab a bit early, I walked over to the historical part of Center City and visited the Federal Reserve Bank of Philadelphia, a place I had never been to before.

Here I am splitting my plate of cancer cells into two different plates giving them more space and new growth media.

The past two weeks have been anything but uneventful as Kennadi and I rapidly work through our project (much faster than Joe and Dr. Bouchard’s expectations).  Looking ahead, we expect to progress in a similar fashion as before with the new primers as Joe and Dr. Bouchard discuss what new project they should have Kennadi and I work on together.