Team:University of Ottawa/21 July 2008

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Today in the lab
Dan
 * Gel of sample 2T
 * <li>For some odd reason all of the DNA stayed in the well... Could be some sort of salt contamination from NEB buffer 1, or a very large autoligation product. I will test the former by doing PCR cleanup and switching to ligation buffer and then run the gel again.
 * Digestion of 1A 0B and both S and D
 * <li> The above DNA fragments were digested with PstI and SphI in Neb buffer 1, enzymes were denatured after an hour
 * <li> PCR cleanup was performed on the digested products, eluted with 50 uL water.
 * Ligation
 * <li> Ligation of the DNA obtained from PCR cleanup was run overnight at 16 C

Matt
 * Miniprep
 * <li>I performed a miniprep of the inoculated PTP2 plasmids and yielded good concentrations for all samples.
 * <li>I then proceeded to digest each sample with NcoI and run them on a gel for confirmation.
 * <li>It appeared as though NcoI only cleaved at one site on the plasmid - linearizing the plasmid. I am going to try to attempt another digestion with EcoRV/EagI which should give two definitive bands - if successful then I will PCR amplify  with SLN1 primers for integration into yeast.

Chris
 * Transformation of Competent Cells
 * <li>Using ligated AtCRE that Dan prepared 22 July 2008, performed a transformation of competent cells according to protocol
 * <li>Performed transformation on a single sample only, as success was not expected. Used LB plates with ampicillin. Incubated against a control plate with no e. coli.
 * <li>Incubated overnight.