One-by-one pENTRY clone generation from HEK293Ta cDNA sample

BP cloning

Material

  • pExpression plasmid sample (eg. pAD-ORF) or PCR product with attB sequencing flanking the desired gene fragment.
  • pDONR plasmid sample (pDONR221, pDONR223, etc.)
  • BP clonase mix II (5x)
  • 96-well PCR plate x 2
  • SOC
  • NEB 5-alpha competent cells
ID Target gene PCR method Conc. (ng/µL) Memo
1 ddH2O - - Negative control
2 MORF4L1 No barcode 14
3 MRFAP1 No barcode 29
4 BCL2L2 No barcode 5.7
5 BIK No barcode 6
6 MRFAP1L1 No barcode 9.7
7 MORF4L1 Fusion PCR barcode cassette 82 AD barcode replicate1
8 MORF4L1 Fusion PCR barcode cassette 78 AD barcode replicate2
9 MRFAP1 Fusion PCR barcode cassette 218 DB barcode replicate1
10 MRFAP1 Fusion PCR barcode cassette 86 DB barcode replicate2

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.
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 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)
  • Plate each sample to LB+Kan plates
  • Culture/select the transformats by incubating the plate, @37C, O/N.
  • interactome/experiments/20191210-3.txt
  • Last modified: 2019/12/11 12:46
  • by dan