Team:Newcastle/Labwork/27 August 2009

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Stochastic Switch team

Summary

Today Goksel did a restriction digest of our plasmid backbone (pSB1AT3) cutting out the mCherry coding sequence. This will prepare the backbone for the PCR products of ara, sac and sspb, which we intend to build as a construct within this plasmid. The sample was run on a gel and, using the transilluminator, the correct band was cut out of the gel. This can be purified tomorrow.

Today we also did a Genomic DNA prep for E.coli. We need this for our PCR reaction in order to get the sspb degradation controller protein. We used the gram negative protocol provided in the kit. Yesterday we set up 2 ON cultures for DH5alpha E.coli:

  • We spun down the cultures to pellet them, and aspirated the media.
  • The pellet was resuspended in lysis solution T
  • For RNA free DNA we added RNAse and incubated for 20 minutes.
  • Proteinase K was then added and the solution incubated in the waterbath for 30 minutes at 55C
  • Then the Lysis solution C was added and was incubated for a further 10 minutes at 55C
  • During this time the column was prepared to the binding column.
  • Ethanol was added to our solution and the bind, wash and elution steps were carried out as the protocol suggested.


[http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/General_Information/na_2100.Par.0001.File.tmp/na_2100.pdf Sigma Genelute Bacterial Genomic DNA kit protocol]

We will run our results on a gel tomorrow as well as analysing them on the nanadrop in order to determine DNA concentration and quality.



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