Team:Brown/Notebook/September16

From 2010.igem.org

Thursday, September 16, 2010

Gel Purification

Followed 'QiaQuick PCR purification kit protocol (pg19 in manual). Eluted in 30ul for higher vol. Put purified digests back in the freezer in TatPTD box

Ligation of purified digest products (LacI, AraC) into opened, purified J13002 plasmid

Rxn A: 15ul total volume

  • 7.5ul ligase buffer (rapid 2x)
  • 1ul T4 DNA ligase
  • 0.5ul vector (J13002)
  • 3ul AraC (RFC25 purified digest insert)
  • 3ul dH2O

Rxn B: 15ul total volume

  • 7.5ul ligase buffer (rapid 2x)
  • 1ul T4 DNA ligase
  • 0.5ul vector (J13002)
  • 3ul LacI (RFC25 purified digest insert)
  • 3ul dH2O

In fridge(4C) overnight

Double digest of tat-linker brick, pBSsk with EcoRI and PstI

Result is to open up bluescript and prepare tat-linker for insertion. The appropriate TF will then be ligated in.

50ul total volume

  • 5ul buffer H (Promega)
  • 3ul DNA
  • 1ul EcoRI
  • 1ul PstI
  • ul H2O

PCR purification/cleanup of EcoPst double digest

Re-eluted samples in 30ul of EB instead of 50 to up concentration

Nanodrop: TatL = 3.5ng/ul | pBS = 21.5ng/ul

Ligation of purified digest products (tat-linker) into open pBS plasmid

15ul total volume

  • 7.5ul ligase buffer (rapid 2x)
  • 1ul T4 ligase
  • 0.5ul pBS vector DNA
  • 6ul TatLinker insert DNA

in 4C overnight (start 4:30PM)

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