Week 6- Cruise Prep and Mass Cloning

This week I was working on a variety of little projects in order tobe ready to mail the mass cloning plates away and to help Suzanne prepare for the sampling cruise that she will be going on next week.  On Monday, Suzanne and I got packing material from Nancy Christie and got dry ice from the primate center. We packed our twelve cloning plates up and sent them off.  We had to make sure that we put enough dry ice in the packaging because we didn't want our sample plates to die because this process of mass cloning is extremely expensive.  We asked for overnight shipping so that we would be sure the dry ice would still be keeping the samples frozen.  After tracking our package, we were informed that our samples reached Maine on Tuesday morning. 
For the rest of the week we tried to get the Strawberry Hill and Cape Mears samples to work for the SIP.  We repeated the SIP 4 times and each time we had nice looking bands but when we went to amplify our DNA we didn't get amplification. We thought at first that it was how we were cleaning the samples but after having cleaned it properly 3 times and it still not working we decided this was likely not the problem. We did six tubes  and combinded them for each site and condition hoping that this would provided more DNA but the problem still persisted. Another possible issue is that the sodium bicarbonate Suzanne used back in May might have degraded and that active organisms had no bicarbonate to integrate into their DNA. This is a possible problem because when we amplified our DNA the 12 C bands amplified but the 13 C bands did not.  This leads us to think that the bicarbonate had degraded before the sample was even taken.  Since these sites were just extra sites and we were just looking at them for further data, we aren't horribly worried although it would be nice to see what these samples have to show us.  Next week we will try the SIP with just the SH-70 samples to see if we see a trend.
I continues to amplify polaribacter in the different primers that Suzanne has been designing for the last month. We currently have a primer for flavor bacteria and polaribacter for pyruvate carboxylase and one for PEPC.  I worked on cleaning these PCRs and cloning them to prepare them for the primate center to be sequenced.  This is important because these will be our standard curves when we go to start qPCR.  Next week I will be extracting alot of RNA preparing for qPCR.