Latest revision as of 03:44, 23 September 2011

PROJECT

Day 11 - Monday, June 27th 2011

Started a restriction digest and gel extraction of our miniprepped DNA. We wanted to see if we could isolate the part that we had taken from the registry and transformed.
Transformed all four types of competent cells that we have made so far with the same positive control. We want to try and get an idea of how competent each batch is.
We noticed our 4 degree cold room was at 21 degrees! We quickly moved everything to another 4 freezer in another lab. We think we were able to save most of the contents, including all of our plates.
Just in case, we began making LB to prepare a new stock of plates.
Transformed a constitutive promoter and several reporter genes for use in our project
Grew bacteria with the Las and Rhl related parts from the registry from our glycerol stocks overnight so that we could miniprep them and extract DNA tomorrow.

Day 12 - Tuesday, June 28th 2011

The cell competency test was a success. All of our plates had colonies, but some had far more than others. Thus we now know to use those cells as much as possible in our transformations.
The four transformations from yesterday also appeared successful, with a number of colonies appearing on each plate.
We miniprepped the overnights we made from the glycerol stock yesterday.
After the miniprep, we tested the samples and found no DNA. We quickly realized the overnight cultures did not appear to contain any growth. This means either our glycerol stock was bad or something went wrong even earlier. We decided the safest thing to do was re-transform all the affected parts.
Started overnight cultures with yesterday’s reporter transformations

Day 13 - Wednesday, June 29th 2011

The new transformations were successful, although some of the plates had very little colonies.
The overnight cultures were cloudy, so we went ahead with our miniprep.
The miniprep resulted in low (10-20ng/uL) DNA concentrations. We still decided we had enough to proceed with the digestion and ligation.
We repeated the miniprep in the afternoon from the same overnight cultures, using a modified procedure for larger volumes. This gave us much better DNA concentrations (70-120 ng/uL).
Started overnight cultures with the transformation plates. We left them in the incubator too long, so it was hard to isolate single colones
Once digestion was complete, we began to ligate a constitutive promoter together with the LasR gene. We’re trying the 3A assembly for now, and we have the DNA from the second miniprep in waiting in case something goes wrong. However, we had to postpone this because we forgot to cut the plasmid backbone!
We also ran out all of our 4 digestion products on a gel, but the bands did not seperate well.

Day 14 - Thursday, June 30th 2011

We miniprepped the overnight cultures of the remaining five parts.
We digested those 5 parts, along with DNA from yesterday’s second miniprep, and plasmid backbones
We ran all the samples out on a gel to make sure everything was cutting well
We began to ligate part combinations together with the 3A assembly, and transformed them with some extra competent cells from one of our advisors. We also have a standard assembly based method prepared in case this fails.
We made new glycerol stocks of all our parts.

Day 15 - Friday, July 1st 2011

After observing that our 3A assembly transformations did not work, we brainstormed possible places where errors may have occurred.
In the meantime, we started 1A standard assembly to see if we will get better results from this method.
We had left over minipreped DNA, which we decided to use for the standard assembly
The rest of the day we digested all the parts and ran them on a gel
We saved the gel for next week to extract and ligate
We figured out the primers that will be necessary to PCR the backbones from the kit so we have our own stock of them.

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 Notebook   <div style="margin: -40px 0px 0px 420px;font:35px helvetica; color:#444444;">    &nbsp; Week 3</div>