Coming to Summer’s End

As the end of summer quickly approaches, I realize that my time for summer research is coming to a close.  I have read countless articles and done countless runs on the real-time.  Yet, as it goes with research, I am still only in the beginning stages of my research.  As I prepare to go back to William and Mary and start another year of school (senior year), I feel that I need to spend some time creating a document that summarizes all of the things that I have read/learned from lab work this summer.  Then, I will be ready to jump back into research and make more progress during the academic year.   While I am ready to go back to school I still find that I am sad to see the wonderful days of summer fading away.

Troubleshooting

I feel like the great majority of research is troubleshooting.  Then, seemingly by accident, something works and the great works of science are born.  I have spent  a great deal of time this week troubleshooting.  My project involves real-time PCR looking at Secreted Ig and Membrane Ig ratios and then comparing these ratios to Pax5 full length versus Pax5delta2 ratios.  Hopefully, in the end we will be able to find a correlation between alternative splicing and an immune response.  But this week, I have realized that my assay is completely worked out for the Secreted/Membrane stuff and by accident, I have a working Paxdelta2 primer set.  Unfortunately, the Pax full length is giving me a real headache.  I am using a SYBR green probe and likely a TaqMan probe would solve all of my problems but when 3/4 of the assay work with the cheaper reagent, it seems pointless to buy a much more expensive probe (which would mean that I had to repeat EVERYTHING with the new probe because it is not acceptable to complete half of a quantitative study using SYBR green and then use TaqMan for the remainder).  But for now I am still troubleshooting, hoping that maybe by chance I will figure out a way to make this little problem and thing of the past.  And all will be well with my project once again.

My BIG mistake

Okay, so research has been going great.  The real-time has been working and my data was coming out beautifully and I was soon going to have finished processing all of my samples…unfortunately, as I’ve experienced many times before, things in science don’t always work out the way we want them to (or think they are).  So, today, I decided to start off my Monday early by coming into lab and setting up a run before my usual lab hours.  As I was setting up, I realized that for the last two months (the entirety of the summer) I have been running my Pax runs with the wrong primer sets (we designed new ones and for some reason I was still using the old ones).  And even though with a cursory glance it looks like my runs were working, when I really started to examine my data, I could see the problem with the primers for full length.  Thus, I have spent today worrying and considering myself an idiot for making such a huge mistake.  After looking at my run (using the proper primers) I realize that everything happens for a reason.  It turns out that the old primers are picking up my isoform much better than the new primers do.  Thus, those runs that I have been doing all summer may not be useless after all.  I am hoping that I will be able to use the CTs for the isoform from the old run and simply repeat the full length runs using the new primers (which are much more efficient). 

[Read more…]

Back to Lab

After a very relaxing weekend celebrating the fourth of July, I am back in lab.  Unfortunately, the reagents that I ordered last week have still not arrived (holidays truly slow down the shipping).  It will be okay though, I have gathered a lot of data over the past few weeks and I am spending the day sorting it out and actually making some graphs.  In addition to this, I am taking notes as I read more literature.  Hopefully by the end of the week I will have a great deal of data and notes to send to my advisor to show her that I haven’t slacked off just because she left town!  Of course, it is the day after a long holiday weekend so I am struggling.  In fact, I was so gung-ho to get back into the swing of things that this morning I went to set up a reaction before realizing that I still had no reagent.  Oh well, I am getting back into the swing of things and I hope that the final two weeks of my research are as productive as the first five weeks. I do know one thing, am certainly glad to be spending my time reading/working indoors because the stifling heat has returned to Williamsburg and being outdoors is unbearable. 

[Read more…]

Pax5 Research

Hello,

So, summer has officially started.  I have been back at school for four weeks now trying to work on my gene expression study.  Unfortunately, due to machine issues, I have produced very little data.  Friday I was able to successfully run two plates, but other than that I have been spending the majority of my time troubleshooting the machine and reading papers.  It has been nice getting to catch up on my reading and in the process I have learned a great deal about developments in the field.  However, I am ready and waiting for the machine to be fixed so that I can start getting usable data.  Instead of simply complaining about dealing with the broken machine, I should mention that troubleshooting has definitely enabled me to gain the skills necessary to obtain reproducible results.  So, I am sitting here in lab (on this very warm first day of summer) waiting for the machine to be fixed…yet again…using my time to read and catch up on my blogging and hoping that soon the machine will be truly fixed.

[Read more…]