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Lab Session 05/08/09

Stochastic Switch Team

Today we needed to do a miniprep of the CinR (BBa_C0077) and CinI (BBa_C0076) biobricks that were put into an overnight culture in the shaking incubator yesterday. There were three cultures for each brick however we discovered that the labels had rubbed off from the tubes, so we decided to do restriction digests and run all 6 samples on a gel separately to determine their identities. As the BioBrick inserts both have similar lengths, we decided to run them on a 1.2 agarose gel to get the best resolution.

For the miniprep:

We followed the miniprep protocol for Prof. Aldridge's lab.

• We spun all 6 samples in 12 eppendorfs to make sure we had at least 3ml. Using 12 eppendorfs also meant that there was only one spin step. (e.g. 1A, 1B, 2A, 2B...)
• After adding solution one to both tubes we resuspended and added the tubes together again resulting in 6 samples. (Added all Bs to As)
• We used the Solution I + RNAse that had been ready made and put in the fridge.
• After spinning with solution III for 20 mins we pipetted the supernatant into a fresh tube and centrifuged again for 5 mins to ensure less protein and cell extracts were in the supernatant.
• After resuspending the final DNA pellet we froze the sample ready for restriction digest on the afternoon.

For the restriction digest:

We had 6 samples and therefore found it easier to make up 7X the mix we needed for the digest, and add 10ul each to 6 labelled eppendorfs, adding our DNA second. This way we did not have to make up the same solution 6 times. (we did 7X to make sure we had enough.) We used Pst1 and EcoRI restriction enzymes making sure that they were the last thing we added to the digest.

In order to make some 1.2 agarose we needed to make up some more 1x TAE from the 50x stock solution.

• We made up 1L using 980ml distilled water and 20ml 50x TAE in a measuring cylinder
• We weighed 2.4g of Agarose and added 190 ml of 1x TAE (we didn't have quite enough left but since have made up some more)