Week eight: touchdown qPCR

I started off this week by running a touchdown qPCR with the archaeal primers to see how similar the numbers were when compared to the qPCR, that way we would know if the we numbers we get with the touchdown qPCR for bacteria are an accurate representation of what we would see with a normal qPCR. Although the numbers for the touchdown qPCR were higher and statically different it was not by a great amount.

Next ran the touchdown qPCR on the bacterial water samples. The water bacterial samples showed nice peaks but they were at a different spot then the standards were at so the DNA was run out on an agarose gel in order to cheek the size of the fragment. The gel revealed that there was a second band. To eliminate this band we tried a second touchdown qPCR at a higher annealing temperature that had previously been showed to produce only one band. This qPCR showed even sharper peaks but at the same early temperature and once again produced two bands. To try to isolate the problem, bacterial samples from many different annealing temperatures were run out on a gel and compared. What the gel revealed was that using an annealing temperature higher then 60°C and lower then 59°C caused the appearance of the second band meaning that it is unlikely that a touchdown qPCR can be used.

This week I also preformed Reverse Transcription PCR or RT-PCR on all of the RNA samples using the Superscript III kit by Invitrogen. Once the RT-PCR was preformed I attempted to amplify the newly created cDNA. I used both primers we had for the archaea. The first primer gave unreadable results because the background in the blanks was so high that it may have masked the low numbers of cDNA. The second primer set showed good amplification in some but there was still noise that may be affecting the lower samples.

I also received pictures of my sampling trip from last week: