Subcloning Protocol
Follow this simple and efficient cut-and-ligate process to transfer a TrueORF insert into any of our PrecisionShuttle destination vectors.
Required Reagents
- TrueORF cDNA clone
- Destination vector
- Sgf I (Asis I) and Mlu I, or Sgf I (Asis I) and Rsr II
- T4 DNA ligase
- Water, nuclease free
-
Digest the TrueORF entry clone:
| Component | Volume |
|---|---|
| 10X restriction buffer | 2 µl |
| Sgf I (10U/µl) | 0.6 µl |
| Mlu I (10U/µl) | 0.6 µl |
| Nuclease-free water | 13.8 µl |
| TrueORF Entry vector (200ng) | 3 µl |
| Total Volume | 20 µl |
Incubate at 37°C for 3 hrs.
- Digest the TrueORF destination vector:
| Component | Volume |
|---|---|
| 10X restriction buffer | 2 µl |
| Sgf I (10U/µl) | 0.6 µl |
| Mlu I (10U/µl) | 0.6 µl |
| Nuclease-free water | 13.8 µl |
| TrueORF Entry vector (200ng) | 3 µl |
| Total Volume | 20 µl |
Note: For the 4% of the clones that have internal Sgf I or Mlu I sites, please use the appropriate combination of restriction sites as recommended by OriGene.
Incubate at 37°C for 3 hrs. Add 0.5 µl antarctic phosphatase (units used according to the manufacturer’s protocol) to the digestion and continue to incubate at 37°C for an additional 30 minutes.
- Purify the digestion using a commercial PCR purification column and elute in 20 µl 10 mM Tris.
-
Set up ligation reaction:
| Component | Volume |
|---|---|
| 10 x T4 DNA ligation buffer | 1 µl |
| T4 DNA ligase (4U/ µl) | 0.75 µl |
| Nuclease-free water | 3.25 µl |
| Digested DNA from Step 1 (ORF clone) | 2 µl |
| Digested DNA from Step 2 (destination vector) | 3 µl |
| Total Volume | 10 µl |
Incubate the ligation reaction at room temperature for 1 hour.
- Transform the ligation reaction into high-efficiency, competent E. coli cells (≥ 1×108 CFU/µg DNA) following the appropriate transformation protocol. Plate the transformants on LB-agar plates supplemented with 100 µg/ml ampicillin for non-Lenti vectors or 34ug/ml chloramphenicol for Lenti vectors.
- Pick at least four colonies for subsequent DNA purification and screening. Amplify and purify the selected clone(s) by growing overnight in liquid LB containing the corresponding antibiotics (ampicillin for non-lenti vectors, chloramphenicol for lenti vectors), then isolating the DNA using standard plasmid purification procedures.
- Confirm the insert by restriction digestion and/or vector primer sequencing using VP1.5 for 5’ end sequencing and XL39 for 3’ end sequencing (non-lenti vectors). A different set of sequencing primers are used for TrueORFs cloned in Lenti vectors; V2.0 as the forward sequencing, LR50 as the reverse sequencing primer.