Week Six: Midterms, hiking, and extractions, oh my!

What a busy week.  We're almost done working directly with our samples, and will be doing lots of data analysis in the upcoming weeks.

On Monday, we worked on purifying our day 0 RNA.  The RNA extraction procedure doesn't distinguish between RNA and DNA and so we need to remove the DNA contamination from our RNA samples. 

On Tuesday, we extracted all the DNA from our day 7 samples and tested the nitrate levels for day 6 and 7.  Because we had 10 different samples (5 conditions in duplicate), it took a long time to get these two things accomplished.

On Wednesday, we extracted and started to purify the RNA samples from day 7.  We had big, fluffy white stuff in samples 1-5 suggesting an excess of detergent.  This is a little unusual though since each sample was done exactly the same.  Mouzhong suggested that it could be because our samples were larger for those samples than the others.  In any case, we had to degrade our excess detergent using TE buffer and sodium acetate and isopropanol to reprecipitate our RNA.  This worked in dissolving our detergents, but we added an excess of TE buffer and had to use large 15ml tubes.  This made it extremely difficult to get our RNA pellets back out of the tubes because they were so tiny at the bottom.  It took a long time to dry the ethanol off the pellets, and we ended up transferring them to a smaller tube to speed up the process.  We left the resuspended RNA (in water) overnight to be purified tomorrow.

On Thursday we purified the difficult samples from Wednesday and quantified all the samples from day 0 and day 7 using the nanodrop and RiboGreen fluorescent dye.  We also ran PCR on our DNA and RNA samples this evening.  Any RNA that contained residual DNA contamination would show amplification (which RNA shouldn't do).  We had a few samples that did show this contamination. So....

On Friday, we purified the contaminated RNA again, to digest the left over DNA.  At lunch, the interns had their midterm presentations during lunch.  The critera was:  one slide, 5 min.  I think my presentation maybe was a bit too introductory in that I included mostly background and the basic information of my experiment, instead of a more detailed report of procedures and graphs of preliminary data.  Oh well, now I know what I can do to improve my final.  After that, in lab we converted our now pure RNA to cDNA.  This is the complimentary strand of DNA to the RNA we started with.  If you remember, RNA contains uracil while DNA contains thymine.  This conversion makes it so that we can use PCR on our RNA via our cDNA. 

On Saturday, a group of us interns went on a hike with Ethan to Mt. Hood.  Whew!  What an exhausting day, but boy were there some pretty views.  I'm not sure when I will be able to move normally again, but it was worth it. :)