Week 5 – Waiting

This week was a little bit slower for the others while we waited to hear back about the sequencing results concerning the DNA fragments we constructed in the previous few weeks. Monday morning we sent out all of the samples from last week to be sequenced. Afterward I prepared an LB/agar solution and put it in the autoclave. Then I spent some time finishing my blog for last week and working on my PowerPoint slide for the midterm presentations this Thursday. After lunch, I took the LB/agar mixture out of the autoclave and put it in the 55^oC water bath. Once the mixture was cooled I poured the LB/agar plates. While I waited for those to set, I worked with the Geneous sequencing program to create the fragment that we are expecting to get back from the Primate Center lab tomorrow (the upstream and downstream regions from the TERTU_2288 gene). When I was done with this the agar plates had still not set up. We realized that I had added too little agar, so I got rid of the plates and then washed the beaker that the agar was mixed in. I guess I know what I will be doing tomorrow! Tuesday morning I worked on finishing my PowerPoint presentation for Thursday’s internship midterm presentations. At noon, all or the interns met and we went over what to do Thursday and did some practice in smaller groups. After lunch I made more LB/agar solution for plating. Then I looked at the sequencing results that came back this morning for the TERTU_2288 gene fragment. Two of the six fragments looked good enough to send back (one looked really good and had no mutations, the other looked good but was shorter than expected for some reason). I transferred the fragment solution and the reverse primer to new tubes and put them in the freezer. Tomorrow morning we will send these to the Primate Center to be sequenced. Before I left, Hiroaki gave me the protocol for preparation of E. coli competent cells before, so I looked that over. Tomorrow we will prepare the competent cells, and then hopefully at the end of this week or the beginning of next week we will get back the sequencing results and be able to use them. Wednesday morning I read parts of a journal article and looked over the protocol for making competent E. coli cells. Afterward Hiroaki showed me the procedure for making competent cells. This took quite a while, so we took a break for lunch part way through while the cells were incubating. Once we were done, I analyzed the sequencing results for the tnbA fragment which had come in earlier today. Unfortunately, all of the fragments had mutations, and were thus unusable for our experiment. Before I left I inoculated six new colonies from the colony isolation agar plate made last week. Thursday I worked on extracting the DNA fragment from the six bacterial colonies inoculated yesterday. Once this was done, I took samples from each and put them in a bag with a sample of the forward sequencing primer. Then I put the bag and the rest of the DNA solutions in the freezer. This morning Hiroaki had found a sequenced Δ1990 fragment from a few years ago, so we moved forward in our experiment with this fragment since we are having trouble finding fragments without mutations for the other genes. As a result, after lunch Hiroaki showed me how to set up the enzyme digestion reaction to remove our fragment (Δ1990) from the T-vector plasmid. This afternoon we only had time for one digestion (the process requires a different digestion enzyme for each end of the fragment) because the reaction has a very long incubation time. In the middle of the afternoon, all of the interns met and we presented on our midterm data and the work we have been doing so far. On Friday Hiroaki showed me how to purify the reaction from yesterday afternoon to remove the enzyme. After purification, he added the second enzyme and buffer set and put the tube in the 37oC water bath to incubate. Incubation takes around four hours, so while we were waiting I had some more time to read and work on my own. At noon all of the interns met for another “brown bag” seminar which discussed policy-related careers that are in science fields. In the afternoon once the digestion reaction from this morning was done incubating, we purified it using agarose gel electrophoresis and a QIAGEN extraction kit. Then we put together the ligation reaction and put it in the 4^oC cooler to incubate over the weekend.