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Probelms with Cre-Recomination


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#1 Trinity

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Posted 07 December 2004 - 03:35 AM

We use the loxP/cre System from Clontech (BD Biosciense, BD Creator) to create our plasmid constructs. We have a Donor Vector (pDNR-Dual Donor Vector) and want to clone our gene into the Acceptor Vector (pLPS-3'EGFP Acceptor Vector). But it does not work!! There are no clones after transformation. And there is no MCS in the Acceptor Vector.
Has anyone else used that system? Does it work?

Thank you!

#2 Trinity

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Posted 16 December 2004 - 07:16 AM

No idea?????

#3 Osiris-gdw

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Posted 27 January 2005 - 01:46 AM

We use the loxP/cre System from Clontech (BD Biosciense, BD Creator) to create our plasmid constructs. We have a Donor Vector (pDNR-Dual Donor Vector) and want to clone our gene into the Acceptor Vector (pLPS-3'EGFP Acceptor Vector). But it does not work!! There are no clones after transformation. And there is no MCS in the Acceptor Vector.
Has anyone else used that system? Does it work?

Thank you!

Hi,
we also use CREATOR with the same Donor- and Acceptor-Vectors...

Yes we also have some problems but we had one positive result with a YFP-Acceptor.
Two hints:

1. The colonies on sucrose-agar grow extremely slow, our positive clones needed 3 days in a 37C incubator to be visible!

2. You can easily insert a MCS in these vectors, just amplify them by PCR out of perhaps EGFP-C1 with primers suitable for LoxP and then use BD In Fusion Cloning Kit...and hope, the Cre-Recombinase will work...
We asked BD for a positive control!

Please post it here if you find better solutions for your problem!

Good luck!

G. :)




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