Personal Interactome
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.