Week 9: Gels and Quantification

I started out the week running some sediment DNA samples through the fluorospectrometer to try to accurately quantify the DNA.  The fluorospectrometer has proved a lot easier to handle ever since we got new chemicals, and all of the samples but two showed readings in the range of the standards.  I also ran a PCR of some samples of different dilutions to see which dilutions we would use for sequencing; after PCR, I ran the samples in a gel, but we could not verify which bands were indicative of the correct base pair when using the 1 kb ladder as a reference.  We ran the samples again side by side with a 100 bp ladder, but were still unsure of which samples were displaying the correct bands.  Therefore, we ran the samples again with what we thought was a cut plasmid which would act as the control for comparison; however, when we ran the samples in gel the cut plasmid did not separate, which indicated that it had not in fact been cut yet.  So we added the enzymes to cut the plasmid and incubated the sample after which we again ran the set of samples through gel.  This time around was a success, and by matching band separation between the cut plasmid and the various samples, we were able to verify which dilutions displayed the correct size of DNA fragments.  On Thursday, I purified two sediment samples with a PCR purification kit (the two samples that had low concentration readings earlier in the week), and then ran those samples through the fluorospectrometer in order to quantify the DNA.  When we looked at the quantification results of all the sediment samples we had ran that week, we realized that we were going to have to extract more DNA in order to attain about 2 µg of DNA, which we were far off from at that point; we are prepping these sediment samples for further quantification using the method recently developed by Smith et al. 2014.