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A 332 base pair mystery and a farewell

As for the last week in lab I amplified and isolated the 332 base pair region in 28S Katablepharis in the Columbia River estuary differs from 28S Katablepharis japonica. This process is just to insure that this frame is real and it is not a misread on the sequence. I'm a little bummed that I didnt get to do real time PCR but thats the nature of experiments sometimes. I will be interested to see the future outcomes of the project.

Longest introduction to the Picture of the Week:

An empty office and an unknown fragment

 Coming to CMOP on Monday was different than usual. The building was quiet and the intern office is empty - except for me. It is a bit lonely without the other interns who I have gotten to know and like the past couple months. I do have much more room to spread my stuff out in the office though:

Final Presentations and still more 28S!

 Wow that last week went really fast. Using the new 28S primers I developed, I finally got the right product for the 28S region. We received the sequences on friday and running through the NCBI Blast program confirmed a match to Katablepharis 28S. Now we have about 2/3 of the 28S sequenceBefore the Primate Center closed on Friday I rushed over some samples to be sequenced with more primers. Hopefully we can get that whole sequence next week.

A new approach to obtaining THE katablepharis 28S

 The jet lag I experienced in North Carolina quickly wore off as I came back to Oregon. I am really excited to be back in the lab. On Monday I designed a primer that is still specific to Katablepharis, but starts in a different region than I had previously attempted amplification. As stated in previous blogs, I was getting partial sequences of Katablepharis 28S. This newly designed primer will hopefully amplify the complete sequence, which will size in at about 3000 base pairs. I am very excited to see the results of the PCR using this new primer.

Going east after extractions

 This week was a short one for me in Beaverton. I had free reign to the PCR machine since many of the graduate students were out on the research cruise in Newport, OR to collect samples. I was able to run ITS and 28S PCR program. This was very nice since the 28S PCR program runs 35 cycles totaling 4 hour and 30 min program!

Primers Primers and more Primers

 Since the primers that Pete and I have been using start from the ITS 2 region, the sequences that we receive only account for about the first 400 base pairs of the 28S sequence. Pete and I developed internal primers so we could possibly obtain the full 28S sequence. After using these primers in PCR we could see that we were not getting the right size fragment. Next week we will try and optimize the PCR program to give us the right product.

More 28S and a trip to OHSU

Continuing on last week's progress, I obtained more 28S sequences. However, since the specific primers start from the ITS 2 region, only a fragment of about 400 base pairs of the 28S region is amplified. The 28S region is approximately 3000 base pairs long. Next week I plan on developing primers within the 28S region to obtain more of the sequence.

A visit to the primate center

 Although it was a short week I managed to make a lot of progress with the 28s region of Katablepharis. The PCR using specific primers showed product so from there I cloned and transformed them into cells. I took the cells and plated them on selective plates with X-gal and ampicillin antibiotics and let them incubate overnight. The following morning I took the white cultures (the blue ones signify an incomplete transformation) and inoculated them overnight. The following day I isolated the plasmid via miniprep and digested it with EcoR1 enzyme.

28s success!

 This week started off with multiple DNA extractions from various locations off the north channel cruise. I ran PCR with those samples along with positive control samples that definitely contained Katablepharis sp. I used the 28s primers Pete Kahn and I designed the previous week. Following the PCR a run on the gel showed that the positive control samples showed 28s amplification. Next week we will continue to perform a cloning and transformation process so that we ultimately can begin to get some sequences for the 28s region!

Finals Week Caught Up With Me

 The cough that I've had since finals week progressed into a case of pneumonia. After a weekend trip with the doctor, I spent the first part of the week in bed trying to rest. I'm glad to say that I'm feeling much better now and was very anxious to get back to work.

On Thursday, Pete, Sheedra, Deirdre, and I took a field trip to collect water samples. In Oregon we took samples at Hammond, Chinook, and near the Rogue Brewery in Astoria. In Washington we took samples at Ilwaco. The weather was beautiful and the sun was out too. (The picture below is from Hammond)


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