Team:University of Ottawa/10 July 2008

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Today in the lab
Dan
 * Gel of S&D&T PCR products
 * <li>The bands I needed were on the gel, they were somewhat close to non specific binding bands, so I took extra caution when cutting them out.
 * <li>After using the gel extraction kit the obtained concentrations were decent.
 * PCR amplification of S&D&T
 * <li>Performed PCR on the S&D&T obtained products, if it works, It would be easy to obtain more DNA and would save the hassle of going back to step 1.
 * Atcre and construct 1 concentrations
 * <li> Both these DNA fragments resulted in very low concentrations following gel extraction. Chris went ahead and used Atcre anyways. The concentration of construct 1 was too low to use.
 * <li> Talked to Cory and realized that AgeI could be used instead of ClaI. That would solve our problem of performing ligation on a 2bp overhang.
 * <li> We ordered new primers today for this.

Looking back
 * <li> After Matt encountered problems with reduced enzymatic activity of restriction enzymes, I decided to redigest and reconfirm

Matt
 * PCR confirmation
 * <li> Tested absorbance and Ran a PCR confirmation of IP producing cells (97 plasmid int. in BY4742 yeast strain) using both wild type BY4742 and H20 as control's.
 * Digestion of PTP2 Ligation Product
 * <li> Another digestion of PTP2 was attempted using EcoRI - sadly I did not get the bands I needed, it seems the PTP2 was not correctly integrated into the Vector.

Chris
 * Ligation of AtCRE
 * <li> ligated AtCRE with 1 uL T4 ligase, 5 uL buffer and 1 uL enzyme.
 * <li> encountered issues with protocol and communication; standard ligation not exactly followed
 * <li> denatured ligase at 65 C for 10 minutes
 * PCR Amplification of AtCRE
 * <li> followed Phusion high fidelity PCR protocol
 * <li> forgot to dilute primers tenfold
 * <li> prepared a 1% gel for confirmation of AtCRE; ran out of time to run gel.