Team:Newcastle/Labwork/2 September 2009
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Revision as of 17:50, 2 September 2009
Contents |
Lab Work - 02/09/09
Promoter Library Sub-Project
Introduction
In yesterday's session, PCR amplifications for the three types of sigA promoter variants was carried out. The amplifications of these three types of promoter variants will give rise to a library of different sigA promoters, hopefully of differing affinities.
In today's session the PCR products will be run on the gel and analysed. If there are signs that the PCR reactions have worked successfully then the PCR products will be purified. If it is apparent that the PCR reactions haven't worked at all then another attempt at PCR shall be made.
Preparing the gel and the DNA gel electrophoresis
The fragments we are expecting to be seen in the gel are roughly 100bp long therefore a fine gel is needed to capture these PCR products. Instead of making up 0.8% agarose gel, 1.5% agarose gel was made up and melted in the way the protocol dictates.
With the gel now set in the tray 6ul (5ul of PCR product and 1ul of loading buffer) of each PCR product was placed into the wells of the gel. The electrophoresis machine was switched on to 90mv and the process allowed to run for 30 minutes.
The resulting gel photograph can be seen here:
At first glance it seems that the PCRs have completely failed but on closer inspection there are little clouds of flourescing material just below the second row of dark bands (caused by the colour bands)in the middle of the lanes. These could well be PCR products; another attempt at gel electrophoresis would clarify this. It is possible that the DNA gel electrophoresis has been left to run too long and additionally the voltage has been set too high. Of course, the white cloudy substance at the bottom of these wells are the primer-dimer complexes.
Stochastic Switch Team
Summary
Today we froze our strains into the TPA collection, by freezing them down in DMSO in the -80 freezers both in the lab as well as duplicates in the stock freezer. Goksel did a digest of the sspb PCR product that was cleaned up yesterday.
Yesterday a PCR was done of CzrA, sacA, ara, and sspb. The products of these were run on a gel, the results of which are shown below. It seemed that there was no PCR product for both the CzrA, and ara, however there is a shadow from all of the wells at around 200bp that could be contamination of PCR product- we are unsure.
Despite inconclusive gel results a PCR product clean up was done on all four reactions. Restriction digests were done on the sac and sspb purified products, which had reasonably clear bands on the gel. These were cut with EcoRI and pstI ready for ligation into the plasmid backbone.
sspB digest and DNA extract
We digested sspB and run the final 70ul of the mix + 10ul of loading buffer on the gel with large wells. We then cut the gel fragment containing our sspB cut with EcoRI and SpeI sites. The DNA of ths sspB insert was then extracted from this gel fragment.
News
Events
- 20 – 21 June 2009 - Europe workshop (London)
- 23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
- 23 October Practice Presentation (Newcastle)
- 23 October T-shirts are ready
- 27 October Practice Presentation (Sunderland)
- 27 October Poster is ready
- 30 October – 2 November 2009 - Jamboree (Boston)
Social Net
- Newcastle iGEM Twitter
- [http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
- [http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]