Team:BYU Provo/Notebook/CholeraDetection/Springexp/Period1/Dailylog

5/1/2013

KK Over the break we did little Lab work. Kelton was in Rexburg, and Clarice and I didn’t have the necessary primers to continue working. Today the primers came! We submitted primers for all the genes that have been cloned into pIG78 with pIG78 for sequencing. (I believe we included the primers for all the genes). We also have primers for working with CRO in the pBAD plasmid. Today our assignment was to make goals and set plans for what we hope to accomplish by the end of the term. Our plans are to be submitted by Friday. Our plans are spelled out in a table we’re printing, but they include understanding (via sequencing) what is ocurring in the plasmid by May 13th, correcting our system by May 29th, and demonstrating that we can induce Lambda into lysis through expression CRO. We will place CRO on the pBAD plasmid with the pBAD promoter (or on pLAT with a pBAD promoter), and the pBAD promoter is induced by arabinose.

KP Today we were trained in Cholera handling safety and we made our plans and goals for a summer full of success.

5/3/13

KP Today Kendall and I did PCR for the first time. Jordan and Clarice showed us how to do it. We did PCR on our E. Coli that has our lysogenic lambda encased within. We also froze down our lambda e. coli samples for future use.

KK Kelton and I worked with Jordan today to PCR amplify the CRO gene from π9907-infected E.Coli. We boiled the E.Coli to use as template and then followed the PCR protocol as outlined. Jordan actually was performed most of the protocol so that we could learn. Our control was E.Coli that had not been infected with lambda.

5/6/13

We ran a gel of our PCR product that we had created over the weekend. This was the first time I had ever done so, so it was fun and interesting! To make the gel, we mix 100 mL of TAE buffer with 1 gram of agar and heat in the microwave. The agar powder needs to completely dissolve. That mixture, with ethidium bromide, is added to the gel dock, and let set for about 20 minutes, or if you set the gel in the fridge it is a little less time. The CRO gene is about 300 base pairs. However, when we ran our PCR product against the ladder and against our negative control, our PCR product matched the control and did NOT match the length that indicates 300 base pairs. So, we know that our PCR reaction failed. It may be because we used a colony that did not include that lambda prophage.

Today we did the PCR reaction to amplify CRO again, but this time we amplified CRO from colonies that had been infected with our three distinct strains of Lambda. We will check our product tomorrow.

KK, KP

5/8/13

We did a few different procedures today. We started out by doing a PCR purification for BI7701, BI7707, and BI23.... It was successful, but our concentration was very low (11.9). We also set up another PCR for BI7701, BI7707, and BI23... It will be done tomorrow. Today we also did a digest of our plasmid and our cro insert.

Having confirmed that our CRO insert was indeed PCR amplified (see picture above; very faint lines about 500 base pairs; wells 1-3 represent three strains of lambda, while 4 is a negative control), we performed a PCR cleanup on ALL 3 of our samples, in an attempt to isolate concentration of CRO insert possible. On the spectrophotometer our A260 reading gave us a concentration of 11.9 ng/microL. Following that we ran a digest of our CRO insert and pLAT plasmid with pBAD promoter using restriction enzymes PST1-HF and ECOR1-HF. Our pLAT sample was a mixture of pIG12 and pIG13, which according to the parts database are the same plasmid, taken from two different colonies. Having set our vector and inserts to digest, we started a low-melt gel with Jordan’s help. Low melt gels follow a slightly different protocol than normal gels, and use a smaller dock. Our dock was broken so our gel didn’t set very well. Because we don’t have time today, tomorrow we’ll run our vector and insert on the low-melt gel to see what happens. KK, KP

5/9/13

Our low melt gel had holes in the bottom of the first few wells, so the already low-concentration plasmid was diluted between 4 wells. Our vector’s well was viable, and the vector stayed in the well. KK, KP

5/10/13

We were able to cut out our vector from the low melt gel, but our insert was not visible. So, today, we made preparations to run our CRO insert on a low melt gel again. I made the low melt gel and set it to cool, and to remain in the fridge over the weekend. Kelton and Clarice performed the PCR cleanup of a second CRO insert PCR reaction that we ran. The PCR was more successful this time - the bands were much more clearly visible, in all three of our Lambda samples (see the photo that Kelton will upload).

Today we did a PCR cleanup on our Cro PCR products. We also set up a slow melt gel so we can try again on Monday to get a cutout of the insert in order to combine our plasmid and insert. We already have the plasmid, we just have to wait on the insert because it wasn’t visible in our first slow melt gel. Here is a picture of our second PCR product run on a 1 kb ladder gel: KK, KP

5/13/13

Today we ran our digested CRO insert on the low melt gel, and under the UV light we saw the band we were looking for. Clarisse excised the band and we performed a ligation reaction according to the protocol and using the vector that we had already run on a low-melt gel last Thursday. After incubating for 30 minutes at room temperature, we performed a transformation into DH5alpha. The selectable marker of pLAT is ampicillin.

We ran our low melt gel with our Cro insert. After 45 minutes, we put the gel under UV light and cut out the insert. We then performed a ligation of our vector and cro insert. After 30 min, we did a transformation and inserted our newly ligated plasmid into DH5alpha.

KK, KP

5/14/13

(Thursday) When we dropped by today two of our plates (out of four) were contaminated. The two contaminated plates, we noticed, were the two plates that were old. The other two plates had a few colonies, but the was not a significantly greater number of colonies in our plasmid + insert + E.Coli plate than there were in our control plasmid + E.Coli plate. Kelton went ahead and streaked the few colonies we had to singles. We can PCR-verify if any of them have the plasmid. Meanwhile I am going to redo the transformation today. I will plate 4 plates: one 50 microL test, one 100 microL test, one 50 microL control, and one 100 microL control.

KK, KP