|
|
| Line 4: |
Line 4: |
| | | | |
| | {| width="100%" | | {| width="100%" |
| - | | colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Cholera Detection May - June Notebook: May 1 - May 12 Daily Log'''</font> | + | | colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Cholera Detection May - June Notebook: July 1 - July 7 Daily Log'''</font> |
| | | | |
| | <br> | | <br> |
| Line 30: |
Line 30: |
| | <font face="Calibri" size="3"> | | <font face="Calibri" size="3"> |
| | | | |
| - | <font size="4"> '''5/1/13''' </font> | + | <font size="4"> '''7/1/13''' </font> |
| | + | |
| | | | |
| - | -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.
| |
| | | | |
| | <br> | | <br> |
| | | | |
| - | <font size="4"> '''5/3/13''' </font> | + | <font size="4"> '''7/3/13''' </font> |
| | | | |
| - | -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.
| |
| | | | |
| | <br> | | <br> |
| | | | |
| - | <font size="4"> '''5/6/13''' </font> | + | <font size="4"> '''7/5/13''' </font> |
| | | | |
| - | -KK 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. This is what our gel looks like: 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, CH, KP |
| | | | |
| - | -KP Today we ran a gel to check our PCR products. Unfortunately it ran farther than it should have and our control came out the same as our test, so we obviously didn’t get what we wanted. Today we started three new PCR’s. BI7701, BI7707, BI23----. We will check them on Wednesday and hopefully we get the results that we want.Our picture.
| + | State of Affairs: |
| | + | |
| | + | Regarding CRO control of lysis in our E.Coli hosts: |
| | + | CRO is inserted into pIG12 and is sequence verified. PIG12 was electroporated into TT9901 and TT9907, which we have verified have the dormant Lambda prophage in their genomes. The electroporation into TT25281 failed. We are plating out TT25281 fresh from a stab, and will electroporate it again. Today, we plated patches of TT9901 and T9907 on LB+amp+arabinose! |
| | + | |
| | + | Regarding Quorum Sensing |
| | + | PCR products of HapA promotor region and CQSS looked good on the gel. We need to redo GFP. |
| | | | |
| | <br> | | <br> |
| | | | |
| - | <font size="4"> '''5/8/13''' </font> | + | <font size="4"> '''7/8/13''' </font> |
| | | | |
| - | -KP 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.
| |
| | | | |
| - | -KK 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.
| |
| | | | |
| | <br> | | <br> |
| | | | |
| - | <font size="4"> '''5/10/13''' </font> | + | <font size="4"> '''7/10/13''' </font> |
| - | | + | |
| - | -KK 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).
| + | |
| | | | |
| - | -KP 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:
| |
| | | | |
| | <br> | | <br> |
"
