Cre-Lox recombination of plasmids - Revision history

ESO wikiadmin at 14:44, 30 May 2025

2025-05-30T14:44:47Z

← Older revision		Revision as of 16:44, 30 May 2025
Line 9:		Line 9:
	** 1 µl (200ng) acceptor vector (e.g. pLPS3’-EGFP)		** 1 µl (200ng) acceptor vector (e.g. pLPS3’-EGFP)
	** 2 µl Cre-recombinase		** 2 µl Cre-recombinase
	* Incubation for ~~45 min~~ at 37° C, meanwhile starting heating-unit		* Incubation for 2h at 37° C, meanwhile starting heating-unit
	* Heat inactivation for 10 min at 70° C and subsequent cooling to room temperature		* 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		* Transformation in ''E.coli'' Nova blue and plating on LB-cam30/sucrose

ESO wikiadmin at 14:44, 30 May 2025

2025-05-30T14:44:33Z

← Older revision		Revision as of 16:44, 30 May 2025
Line 1:		Line 1:
			{{Protokoll-Mix\|titel=Materials\|Inhalt=Donorvector (pDNR-Dual with your construct) Acceptor vector (pGEX4T1)<br>Cre-buffer, 10x<br>Cre-Recombinase [10 U/µl] Aqua bidest<br>Heating-unit, 70° C<br>-> Subsequent method: Transformation}}

	== Procedure ==		== Procedure ==

ESO wikiadmin: Created page with "== 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..."

2025-05-30T14:43:30Z

Created page with "== 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..."

New page

== 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

# 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)
# 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
# restriction digest in comparison to the used donor vector and the used empty acceptor vector