Team:University of Ottawa/10 July 2008

From 2008.igem.org

Contents

Today in the lab

Dan

Gel of S&D&T PCR products
  • 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.
  • After using the gel extraction kit the obtained concentrations were decent.
    • PCR amplification of S&D&T
  • 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
  • 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.
  • 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.
  • We ordered new primers today for this.

    • Looking back

  • After Matt encountered problems with reduced enzymatic activity of restriction enzymes, I decided to redigest and reconfirm

    • Matt

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