Week Four: qPCR problems

This week I had some problems with the qPCR I was preforming. The 1^st one contained all the samples we had extracted from the sampling trip last week.  While all the samples showed nice peaks with a good quantity of the gene many of them had their peaks a degree off of the optimum temperature.  To try to rectify this we attempted the qPCR with a shorter primer for the AmoA gene.  This qPCR had perfect peaks for all of the samples but sadly also had peaks in the blanks.  Since this had not happened before we believed the contamination was in the new primer set so a third PCR was preformed to test these new primers against the old ones along with a spare set of the short sequence AmoA gene primer. The test revealed that both of the short set AmoA primer had contamination while the old primer set did not. We ordered more of the short sequence primer so that the experiment can be repeated.

While waiting for the primer set for the Archaea, I ran a qPCR on a few of the filter samples for the bacterial AmoA gene. This qPCR  also did not work but after a quick qPCR lesson from Mouzhong, we realized that this may be because of a dilution problem so we will evaluate this and see where to go from there.

That was all I got done in the lab this week because of the forth of July and the trip to the National Park to see the pictographs on Friday. Although both of those were amazingly enjoyable.