Material

  • pDEST plasmid sample (eg. pDEST-AD, pDEST-DB, pDEST-DHFR …)
  • pENTRY plasmid sample
  • LR clonase mix II (5x)
  • 96-well PCR plate x 2

Protocol

1 Substrate preparation
  • Prepare 60ng/ul solution of pDEST plasmids.
  • Prepare 60ng/ul solution of pENTRY plasmids.
  • Prepare 2 96-well PCR plates indicating name, sample, and date.
  • Mix 2ul each of the DNA as follows into one PCR plate.
  • Aliquot 1ul of the DNA mix to the other PCR plate.
2 LR reaction
  • Prepare Ice with a tube labled “LR”
  • Thaw LR clonase mix II on ice (~2mins).
  • Breifly vortex (1sec x 2), and centrifuge.
  • Take Xul of clonase mix from tube and aliquot to the labled tube.
Check Reagent 1 reaction (µL) _ _ _ _ reaction (µL) Final concentration
ddH2O 0.6 -
5x LR clonase mix II 0.4 2x
Total volume (µL) 1.0 2x
  • Return enzyme mix to -80C freezer.
  • Add X µl of ddH2O to the 5x LR clonase mix to make a 2x LR clonase mix.
  • Pipette well to mix the enzyme mix until the solution is completely mixed.
  • Add 1 µl of the 2x enzyme mix to each of the the DNA mix prepared in PCR plate.
  • Incubate @25C on a PCR machine O/N (>16h)
3 Transformation
  • Prepare the reaction mix and competent cells on ice.
  • For each sample, add 10µl of homebrew competent NEB5-α cells.
  • Incubate on ice for 20mins
  • Heatshock on PCR machine @42C for 30secs
  • Incubate on ice for 2mins
  • Outgrowth by adding 100ul S.O.C. and incubate @37C for 1 hour (static)
  • Spot 5 µL of each sample to LB+Amp plates
  • Culture/select the transformats by incubating the plate, @37C, O/N.

Dan Yamamoto-Evans 2019/11/18 17:46

  • interactome/lr.txt
  • Last modified: 2019/12/02 10:17
  • by dan