Team:Newcastle/Labwork/27 August 2009

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Contents 1 Stochastic Switch team 1.1 Summary 1.2 Restriction digest 1.2.1 Running the gel

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.

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.

Restriction digest

Before we start using pSB1AT3+mCherry from the midipreps we diluted the tube with 100ul of water. We then followed the steps to cut the plasmid from EcoRI and SpeI sites to remove the biobrick. The plasmid backbone will be used for our parts later on.

• 15ul of water
• 4ul of fast digest buffer
• 15ul of DNA(pSB1AT3)
• 3ul of fast digest EcoRI
• 3ul of fast digest SpeI

After mixing everything we added 50ul of the loading buffer to make the final volume 50ul.

Running the gel

We prepared the gel with large wells to be able t load 50ul. We used 10ul of DNA ladder.

We then cut the gel fragment containing the plasmid backbone.