Haynes Lab:Notebook/TB Biosensor/2012/07/16

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Didn't get any colonies from yesterday's transformations. Dr. Haynes suggests cloning a smaller insert into the large crtE,I,B backbone, rather than cloning the >3kb crtEIB insert into a 2.2kb backbone.

Cut 25ul of crtEIB with EcoR1 and Xba1. Expect a 5.2-5.5kb piece

Cut 25ul of the Lacp with EcoR1 and Spe1. Expect a 2.2kb (backbone) and 200bp (promoter) piece.

Ran the whole reaction on a gel and saw the crtEIB piece and the Lacp backbone but not the 200bp promoter piece. I was dealing with really low yields already (the miniprep was 15ng/ul) so I cut out a piece of the gel where I expected the band to be and purified it. I got a negative spec.

I recut 25ul of the Lacp, purified it using the Zymo clean and concentrator kit, and spec'd. Then I dephosphorylated and ligated with EcoR1