Week Four: (July 12 - July 16, 2010) - Final Extractions (For Now....)

7/12/10:  Today was filled with six extractions, a PCR, and several gels.  In the morning I extracted four samples all together and finished in about four hours (I’m getting a lot better at this now!)  I also ran the gel for the duplex PCR that I did on Thursday, but the signals on the gel were too good, meaning I either had to much of the sample or I needed to dilute the PCR products.  I re-ran the gel with less of the sample, but I still had a very strong signal, so Dr. Zuber had me re-run the PCR (18S and NITS). This time I lowered the annealing temperature, or lowest temperature, of the PCR because it is supposed to make the PCR work better, so I’ll find out tomorrow.

7/13/10:  I got my results of the PCR with the lowered annealing temperature, but it didn’t work that well.  The positives, or samples that will always have a signal, did not show too strong of a signal but the actual samples hardly had any signals on them.  To see if it was just the samples, Dr. Zuber had me do the duplex PCR with two new samples, but I got the same results as before.  I also finished the last two extractions (hooray!) and ran all of the extractions on a gel to see if the extracted DNA/RNA is usable.

7/14/10:  Now that I’m done with the extractions it felt like I had a lot of free time today.  After analyzing the duplex 18S and NITS PCR results with Dr. Zuber, we decided to run an 18S and NITS PCR separately then run them on the same gel to compare the results.  The normal protocol calls for 30 cycles of alternating high and low temperatures for the DNA polymerase to happen, but I lowered it to 20 cycles to see if we might get better results of the PCR.    Using 20 cycles will give us a quantitative data analysis of how much Myrionecta is actually in the sample because using 30 cycles could possibly be amplifying the DNA too much.  I will find out tomorrow when I run the gel, but hopefully it worked.

7/15/10:  I got back the results of the NITS/18S PCR with 20 cycles, however the signals were not strong enough on both the positives and the samples, so tomorrow I will do this PCR again except with 25 cycles.  Dr. Herfort also came back from her three day vacation so I had a brief meeting with her and Dr. Zuber about what I have done over the past few days and what I need to do for future work.  Since Vikki will be gone for almost three weeks on the July/August Cruise I will most likely be learning how to do single cell PCR and cloning.  I also ran an NITS PCR for all of the samples I hadn’t done yet as well as for two samples that showed no signal.  Dr. Herfort believes that there is a lot of dissolved particulates in the DNA/RNA sample I’m using, so I had to dilute the samples to separate the DNA/RNA and the dissolved particulates better.  My results for the PCR came back as expected; two new bottom samples showing no signal and the dilutions showing a Myrionecta signal. To make sure our technique is working properly I will run a negative (sample with no Myrionecta) PCR tomorrow.

7/16/10:  I didn’t do a lot of lab work today, as it was mostly spent talking with Dr. Herfort about my upcoming presentation/poster for the ASE Symposium on the 20th of August.  For time purposes and not wanting to rush myself I started prepping for my presentation, so I only ran two PCRs and one gel today.  I ran the 18S and NITS PCR with 25 cycles and then a dilution NITS PCR of the two previous samples mentioned yesterday.  The results for that PCR came back nicely - the negative showed no Myrionecta, and one of the samples showed a signal for Myrionecta, as was expected.  On Monday I plan to work more on my presentation, so again there will be minimal lab work done.

Thanks for reading this week, I’ll post later!

~ Deirdre