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| | {| width="100%" | | {| width="100%" |
| - | | colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Cholera Detection May - June Notebook: May 27 - June 9 Daily Log'''</font> | + | | colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Cholera Detection May - June Notebook: June 10 - June 23 Daily Log'''</font> |
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| | + | We ran our PCR product on a gel - this was the product of the PCR reaction with the new, correct, CRO primers. Despite this, our gel showed one, thick band at the bottom of the gel, but no 300 base pair band. We streaked out TT9901 and TT9907 and TT25281 fresh from the stabs to use as template on our next attempt, and Dr. Grose is going to do it with us. |
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| | <font size="4"> '''6/12/13''' </font> | | <font size="4"> '''6/12/13''' </font> |
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| - | -KP KK Today we plated TT9907 and TT9901. We plated them on LB arabinose/amp/x-gal plates. We plated different concentrations of TT9907 and TT9901 on each of the plates. We added 50, 100, and 300 microliters onto each plate. We will check the plates tomorrow hopefully to find plaques showing that arabinose triggered lysis of our lambda. We unfortunately didn't get any growth of our TT25821, so we streaked another plate from our original stab sample and we will try to re-electroporate TT23821 on Friday.
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| - | NO! Our work over these past few weeks reached its climax today ... we checked our plates, but no dice. No plaques, that is. Lambda was not induced out of lysogeny. We are asking ourselves these questions: Was CRO not actually ligated into our vector? Are our plates not good (we used year old plates)? CRO, when overexpressed, downregulates itself; is there too high a concentration of CRO? Or is it possible that CRO alone isn't a sufficient factor to induce lysis. Dr. Grose talked with us, and told us she had our sequencing!! The results were depressing but revelatory: the sequence for CRO is not in our vector! So, on Monday we will begin the cloning process again. Today we set up a PCR reaction to amplify CRO from strains TT9901 and TT9907 template DNA.
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| | <font size="4"> '''6/17/13''' </font> | | <font size="4"> '''6/17/13''' </font> |
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| - | -KK KP Our PCR product was consistent with every other attempt to amplify CRO: it failed. Consistency is only a virtue if you're not a screw-up :) Anyway, our gel showed a faint band at the 300 base pair level, but too faint to be considered successful. We considered a list of reasons why our PCR reaction is failing and discussed them with Dr. Grose:
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| - | We boiled our template DNA for 10 minutes, but Dr. Grose recommends 5 minutes
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| - | We added 1 microliter of template; Dr. Grose likes to add 2 microliters.
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| - | We did not use a master mix to combine our reagents with our template DNA. We added each reagent individually to each tube. Because we are pipetting such small volumes, there is a lot of room for error this way.
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| - | The Phusion program we used was set to an annealing temperature that was 1 degree too low for Phusion.
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| - | We did the PCR reaction one more time with Dr. Grose. Tomorrow we expect to see an enormous, fat, band!
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| | <font size="4"> '''6/19/13''' </font> | | <font size="4"> '''6/19/13''' </font> |
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| - | Regarding our PCR attempt yesterday, we did see a thick fat band ... at the bottom of the gel! Once again our PCR failed. This has led us to propose several reasons why we can't PCR amplify CRO: 1) the Lambda prophage isn't actually in TT 9901 or TT9907, or 2) our primers aren’t functioning to amplify CRO. We streaked a few plates of TT9901 and TT9907 to subject to UV light. If we see plaques, then we will know Lambda has been induced. Meanwhile Dr. Grose is going to check that our primers are the correct ones.
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| - | After a little investigation, it turns out our primers were NOT for the CRO gene in Lambda, but for the CRO gene in another phage. Dr. Grose immediately ordered another set of primers, which arrived this morning! With the correct primers to amplify CRO, we performed the PCR protocol again today. Also, we set up an overnight of E.Coli with pIG12 to purify the plasmid tomorrow. We'll use pIG12 as our vector.
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| - | Our community project will be writing a children's book on synthetic biology. I wrote a story line which was distributed among all the team members; with their ideas I'm going to make changes and contact Redge Ballard, our illustrator. Since we hope to do our community project in one month, hopefully he can illustrate the story in such a short time!
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