Materials

Most of the materials can be obtained as a kit from Invitrogen:

For instance E. coli Expression System (with GATEWAY Technology) (order number: 11823-010 / 20 rxns)

Enzymes

  • LR Clonase enzyme mix (including LR Reaction buffer)
  • (Invitrogen - 11791-019 - 20 rxns)
  • Proteinase K solution (2 mg/ml)

TE buffer

  • 10 mM Tris-HCl pH 8.0
  • 1 mM EDTA

Procedure

1. Prepare the LR Reaction mix at room temperature as described below. The LR Clonase enzyme mix should be added last.
1 - 10 ml   Entry Clone (100 - 300 ng)
2 ml   linearized Destination vector (150 ng/ml)
4 ml   LR Reaction buffer
4 ml   LR Clonase enzyme mix

TE buffer (to a total volume of 20 ml)
2. Incubate for 60 min at 25°C.
3. Add 2 ml Proteinase K solution and incubate for 10 min at 37°C.
4. Mix 1 ml of LR Reaction mix with 50 ml competent DH5a cells (for the preparation see the appropriate protocol).
5. Follow the protocol for the transformation of plasmid DNA to chemically competent E. coli cells (see protocol database).
6. Plate 10 and 100 ml of the final solution on LB agar plates containing the appropriate antibiotic (ampicillin for Invitrogen's destination vectors).
7. Incubate plates overnight at 37°C.

Remarks

If not yet done, linearize the Destination vector within the attR cassette avoiding the ccdB gene.

Reference

GATEWAY Cloning Technology Instruction Manual Version 1 (Life Technologies)