Week16 9/26/10-10/2/10

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Latest revision as of 23:18, 16 October 2010

Week16 Highlights

We continued work with Cp-LacPi-GFP constructs, but failed to get Cp-LacPi parts based on our gels. We also realized that our pMAL primers were incorrect so we placed an order for new primers. We completed site directed mutagenesis of CHS3.

9/26/10

Upon taking out the cultures, Kevin realized that he and Timi had put the wrong antibiotic into the incubations last night. He came back later in the day to reinoculate, but this time with Kan antibiotic!

Ran gels for the digestions --> no band was less than 1000 bp (Cp-LacPi1 parts should be ~90bp and Cp-LacPi2 parts should be ~235bp)

Is it possible that the digestions could have failed? Maybe you accidentally enabled heated lid?

The files are cplacpiscreengel1(9262010) and cplacpiscreengel2(9262010), cplacpiscreengel2overexposed(9262010) if you want to take a look. (REMOVE THIS LATER)

The bands in the gel were also a little wavy. According to Professor Mordacq, this can happen when the agarose is not properly melted all the way.

Innoculated the CP-LacPi-GFP parts again.

9/27/10

Inserted LacP-RBS into Tet plasmid --> transformed into ChiA knockouts

Kevin went in between classes to spin down the incubations from last night.

Timi picked up from there and miniprepped the samples and then digested them.

9/28/10

pMAL - realized primers are bad --> reordering correct ones

Timi went in early to do 20 uL digests of the CP-LacPI assemblies. BUT she forgot to put in restriction enzymes. FAIL.

Tried Cp-LacPi digestions using more DNA --> still too difficult to visualize the small inserts

This could be several things (1) We just don't have any positive inserts -- which is a possibility given several unexplainable bands above 1000 bp. (2) We don't have enough DNA in the gels to properly visualize it. (3) Something with the gels/apparatus/procedure itself.

9/29/10

Site-directed mutagenesis: Amplification and DpnI digestion completed.

Ragan had some trouble with the TBE buffer. We are looking into what went wrong with it. Sean?

9/30/10

Transformation for Site-Directed mutagenesis completed.

At the lab meeting this morning, Timi discovered that Kevin had been looking for an incorrectly-sized band in the gels. They went back through the photos of gels later in the day, but were inconclusive. They are going to back-track a little bit to figure out if any of the colonies that they had picked were actually positive.

10/1/10

The transformation resulted in a good number of colonies for site-directed mutagenesis. 10 liquid cultures were made.

Timi went in early to pour a big and small gel. BUT she forgot to put in the combs. FAIL.

Kevin repoured the gels and ran the digested DNA on it. However, he forgot to run the digested parts on it as well, as the advisors had suggested.

10/2/10

Timi and Kevin put in 15 overnight inculations of 31-GFP, 32-GFP and 21-GFP.

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 ===Week16 Highlights===
+We continued work with Cp-LacPi-GFP constructs, but failed to get Cp-LacPi parts based on our gels. We also realized that our pMAL primers were incorrect so we placed an order for new primers. We completed site directed mutagenesis of CHS3.
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 Upon taking out the cultures, Kevin realized that he and Timi had put the wrong antibiotic into the incubations last night. He came back later in the day to reinoculate, but this time with Kan antibiotic!
-EDIT THIS:
+Ran gels for the digestions --> no band was less than 1000 bp (Cp-LacPi1 parts should be ~90bp and Cp-LacPi2 parts should be ~235bp)
-So the gels I ran today for the digestions didn't turn out. At least no band is less than 1000 bp. Our Cp-Lacpi parts should either be 90 bp for CP(x) - LacPi1 [might be inherently difficult to see] and 235 bp for CP(x) - LacPi2, for reference. Just to do a little troubleshooting, is it possible that the digestions could have failed? Maybe you accidentally enabled heated lid? I'll try to catch Mordacq after my 3 p.m. class to see what he has to say. The files are cplacpiscreengel1(9262010) and cplacpiscreengel2(9262010), cplacpiscreengel2overexposed(9262010) if you want to take a look.
-Also, the bands in the gel were a little wavy. According to Mordacq, this can happen when the agarose is not properly melted all the way. I usually microwave for 1:30. I'll let it go for 30 seconds, then quickly swish it around, and put it back for the remaining 1:00 so it boils all the way. This is more important when not using low melting point agarose.
+Is it possible that the digestions could have failed? Maybe you accidentally enabled heated lid?
-Finally, I innoculated the CP-LacPi-GFP parts again today around 7:00. I'll pop them in the centrifuge after sys phys to let them grow for a good amount of time and leave them in there for when you get in lab at 2:00.
+'''The files are cplacpiscreengel1(9262010) and cplacpiscreengel2(9262010), cplacpiscreengel2overexposed(9262010) if you want to take a look.''' (REMOVE THIS LATER)
-I'll also prepare the two parts for sequencing from the last gel and give them to Mordacq at 3:00.
+The bands in the gel were also a little wavy. According to Professor Mordacq, this can happen when the agarose is not properly melted all the way.
-Also, here's what we need to get done in the near future...
+Innoculated the CP-LacPi-GFP parts again.
-- make alkaline miniprep lysis, resuspension and neutralization buffer (need to ask Mordacq where glucose is and how to prepare 1% SDS)
-- research plate reader protocols for plate reader exps
-- fill out project notebook with what we've been working on
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 ===9/27/10===
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 ===9/28/10===
-pMAL - realized primers are bad, reordering
+pMAL - realized primers are bad --> reordering correct ones
 Timi went in early to do 20 uL digests of the CP-LacPI assemblies. BUT she forgot to put in restriction enzymes. FAIL.
-EDIT:
+Tried Cp-LacPi digestions using more DNA --> still too difficult to visualize the small inserts
-Still, Kevin wanted to send you the results of the gels.
-Unfortunately, even with digesting more DNA, it is still too difficult to visualize the small inserts. This can be several things (1) We just don't have any positive inserts -- which is a possibility given several unexplainable bands above 1000 bp. (2) We don't have enough DNA in the gels to properly visualize it. (3) Something with the gels/apparatus/procedure itself.
-As far as controlling for DNA run on the gels, we will have to be careful loading the DNA into the gels (given the large volume, there were a few wells where I lost DNA during the transfer) as well as the volumes for our digestions (some of the digestions were not 20 µl -- this can be from liquid evaporating, but since the volumes were inconsistent it can also be pipeting error). Just some things to be aware of.
-In the meantime, we should ask Mordacq what he thinks our next step should be -- whether we want to try again or try another method of screening. We should also double check we're doing the screening process correctly by digesting and running the CP-LACPI-GFP assemblies you just miniprepped. It will be much easier to see the inserts on a gel.
+*This could be several things (1) We just don't have any positive inserts -- which is a possibility given several unexplainable bands above 1000 bp. (2) We don't have enough DNA in the gels to properly visualize it. (3) Something with the gels/apparatus/procedure itself.
-I'm going to send some things in for sequencing tomorrow morning so I may try to digest before class or from 11 a.m. - 1 p.m. Would you be able to run and image a gel tomorrow later afternoon/evening? Not sure if you have Jumpstart, so just let me know.
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