Cre-Lox recombination of plasmids

Materials

Donorvector (pDNR-Dual with your construct) Acceptor vector (pGEX4T1)
Cre-buffer, 10x
Cre-Recombinase [10 U/µl] Aqua bidest
Heating-unit, 70° C
-> Subsequent method: Transformation

Procedure

• Dilute Cre-recombinase [10 U/µl] with 1x Cre-recombinase-buffer 1:10 [1 U/µl]
• Recombination reaction contains:
  • 1 µl 10x Cre-recombination-buffer 6µl aqua bidest.
  • 1 µl (100ng) donor vector (e.g. pDNR-dual gene X)
  • 1 µl (200ng) acceptor vector (e.g. pLPS3’-EGFP)
  • 2 µl Cre-recombinase
• Incubation for 45 min at 37° C, meanwhile starting heating-unit
• Heat inactivation for 10 min at 70° C and subsequent cooling to room temperature
• Transformation in E.coli Nova blue and plating on LB-cam30/sucrose
• Check clones by

1. plating on a further selective medium if the acceptor vector has such a resistance cassette (e.g. LB KanR in the case of pLPS-3’EGFP)
2. overall size of the isolated plasmids in comparison to the used donor vector plasmid, the used empty acceptor vector plasmid, and a third control plasmid of comparable size as the expected correct clone
3. restriction digest in comparison to the used donor vector and the used empty acceptor vector