BP cloning of PCR amplified HEK293T cDNA

Material

  • pDONR plasmid sample (pDONR221, pDONR223, etc.)
  • BP clonase mix II (5x)
  • 96-well PCR plate x 2
  • SOC
  • NEB 5-alpha competent cells (Homebrew)

Protocol

1 Substrate preparation
  • Prepare 6-60ng/ul solution of attB PCR sample/ plasmids.
  • Prepare 2 96-well PCR plates indicating name, sample, and date.
  • Add 2ul of the DNA as follows into one PCR plate.
pDONR PCR sample Date prepared Memo
pDONR221 PI 21 20191125DEY MORF4L1 amplified sample (PCR purified)
pDONR221 PI 22 20191125DEY MRFAP1 amplified sample (PCR purified)
pDONR221 PI 23 20191125DEY BCL2L2 amplified sample (PCR purified)
pDONR221 PI 24 20191125DEY BIK amplified sample (PCR purified)
pDONR221 PI 25 20191125DEY MRFAP1L1 amplified sample (PCR purified)
2 BP reaction
  • Prepare Ice with a tube labled “BP”
  • Thaw BP 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 Stock concentration Final concentration Volume per 1 reaction (µL) Volume per _ _ _ _ _ _ _ reaction (µL)
BP clonase mix II 5x 2x 0.4
pDONR plasmid 60 ng/µL
ddH2O - - Up to 1.0
  • Return enzyme mix to -80C freezer.
  • Add X µl of ddH2O and pDONR plasmid to the 5x BP clonase mix to make a 2x BP clonase master mix.
  • Pipette well to mix the enzyme mix until the solution is completely mixed.
  • Add 2 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 40vµl of homebrew competent NEB5-α cells.
  • Incubate on ice for 20 mins
  • Heatshock on PCR machine @42C for 30secs
  • Incubate on ice for 2mins
  • Outgrowth by adding 400 ul S.O.C. and incubate @37C for 1 hour
  • Centrifuge, plate whole sample to LB+Kan plates
  • Culture/select the transformats by incubating the plate, @37C, O/N.
Check Reagent Stock concentration Final concentration Volume per 1 reaction (µL) Volume per _ _ _ _ _ _ _ reaction (µL)
NEB 5-alpha competent cells - - 10
SOC medium - - 100

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

  • interactome/experiments/20191125-2.txt
  • Last modified: 2019/11/25 17:41
  • by dan