Team:WashU/Week5

From 2012.igem.org

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We digested our new promoter, J23119, using the biobrick protocol (with a slight modification - we used NEBuffer 4 instead of NEBuffer 2, since the enzymes we were cutting with, E and S, also have 100% activity in NEBuffer 4) and ran a gel to ensure that we had pure promoter. We accidentally grew up the promoter in both plain LB and LB + amp, yet still had colonies in both media, and thus decided to run two digests to ascertain that we have our promoter in both cultures. The gel is shown below: <br><br>
We digested our new promoter, J23119, using the biobrick protocol (with a slight modification - we used NEBuffer 4 instead of NEBuffer 2, since the enzymes we were cutting with, E and S, also have 100% activity in NEBuffer 4) and ran a gel to ensure that we had pure promoter. We accidentally grew up the promoter in both plain LB and LB + amp, yet still had colonies in both media, and thus decided to run two digests to ascertain that we have our promoter in both cultures. The gel is shown below: <br><br>
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<div align = "center">
https://lh3.googleusercontent.com/-CK3osVLJrOo/T-iq423PuqI/AAAAAAAAALU/GNmsLsVqnCA/s400/digest1.jpg
https://lh3.googleusercontent.com/-CK3osVLJrOo/T-iq423PuqI/AAAAAAAAALU/GNmsLsVqnCA/s400/digest1.jpg
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<br>
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<div align = "left">
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Unfortunately, the digest did not seem to work. We attempted to digest the same DNA once more, using NEBuffer 2 instead of NEBuffer 4, and ran a second gel [SHOW GEL BELOW]
Unfortunately, the digest did not seem to work. We attempted to digest the same DNA once more, using NEBuffer 2 instead of NEBuffer 4, and ran a second gel [SHOW GEL BELOW]
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<br>
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<div align = "center">
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https://lh6.googleusercontent.com/-G7Ey3-_cpos/T-i8042yNAI/AAAAAAAAALs/12IEso8KhCk/s400/digest2pic2.jpg
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<br><br>
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<div align = "left">
We have also decided to use PCR to amplify our DNA, as we are getting extremely low yields from our gel extractions. Thus, we have ordered primers.  
We have also decided to use PCR to amplify our DNA, as we are getting extremely low yields from our gel extractions. Thus, we have ordered primers.  

Revision as of 19:35, 25 June 2012



Monday, June 25

YLC

At the end of last week we ran a gel of the digests from the fluorescent proteins and extracted DNA from the gels. At the beginning of this week, we used the nanodrop to see how much DNA we had retrieved from the gel. The nanodrop revealed barely imperceptible maxima to indicate that we had almost undetectable amounts of DNA by the nanodrop. In fairness, the bands were tiny to begin with, leading to our poor results. Our mentors assured us that we probably had enough DNA for a ligation, so we plan to run that later on in the day. [MORE ON LIGATION]

We digested our new promoter, J23119, using the biobrick protocol (with a slight modification - we used NEBuffer 4 instead of NEBuffer 2, since the enzymes we were cutting with, E and S, also have 100% activity in NEBuffer 4) and ran a gel to ensure that we had pure promoter. We accidentally grew up the promoter in both plain LB and LB + amp, yet still had colonies in both media, and thus decided to run two digests to ascertain that we have our promoter in both cultures. The gel is shown below:

digest1.jpg

Unfortunately, the digest did not seem to work. We attempted to digest the same DNA once more, using NEBuffer 2 instead of NEBuffer 4, and ran a second gel [SHOW GEL BELOW]

digest2pic2.jpg

We have also decided to use PCR to amplify our DNA, as we are getting extremely low yields from our gel extractions. Thus, we have ordered primers.


Website

The weekly logs were revised and updated today.

Tuesday, June 26

Wednesday, June 27

Thursday, June 28

Friday, June 29

Retrieved from "http://2012.igem.org/Team:WashU/Week5"