Team:University of Ottawa/16 July 2008

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Today on the Lab
Tammy
 * Innoculation of transformed X10 E.coli + pDR197::AtCKX2 in LB + AMP.
 * <li>For each dilution (PURE, 1:10, 1:100), 2 colonies were chosen to innoculate in 3 ml LM + AMP.
 * <li>Control is LB + AMP only.
 * <li>Finished Innoculation at 3:oo pm

Chris
 * Gel Extraction of AtCRE
 * <li> prepared a 0.8% gel and ran the AtCRE sample for 40 minutes at 90 V
 * <li> expected bands appeared and the desired one was excised
 * <li> gel extraction was performed on the excised sample successfully
 * Determining AtCRE Concentration
 * <li> measured the absorbancy of AtCRE, resulting in invalid data. It was determined that the blank was performed incorrectly, such that the machine was not zeroed properly.
 * <li> the absorbancy of AtCRE was remeasured using a new blank at a 1:10 dilution. The resulting absorbancy was 0.3113 at 260 nm, giving a concentration of about 150 ng/ul.
 * Ligation of AtCRE
 * <li> AtCRE was ligated with 1 ul ligase, 1 ul ATP, 2 ul DNA template, 1 uL buffer and 5 ul water
 * <li> the sample was incubated at 16 C overnight

Matt
 * Glycerol Stock
 * <li> The BY4742 cells int 597/598 were inoculated once again for glycerol stock tomorrow - this time I sealed the tubes with the parafin.
 * Digestion
 * <li> Digestion confirmation of PTP2 with NcoI after Gel extraction was successful with correct band sizes confirming that I have the PTP2 product.
 * <li>A PCR cleanup was performed on the digestion product of PTP2 digested with BamHI + xhoI, concentrations were low but not low enough that a ligation cannot be performed.
 * <li>I still have some pSSA42 digestion product from last time the digestion was performed that I can use for this.
 * Ligation
 * <li> A ligation was then performed using PTP2 insert in pSSA42 vector using 3:1 mol ratio - I decided to use a little more insert than suggested to ensure success of the reaction.
 * <li> Dan was thinking it would probably be easier if the ligation was spiked tomorrow morning with more ATP.

Dan
 * Overnight PCR of 0B at 35 cycles
 * <li> Was unsucessfull
 * PCR of 0B at 35 cycles
 * <li>Was redone and did not work again,
 * <li> My conclusion is that the higher primer concentration is inhibiting it.