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Osiris-gdw's Content

There have been 2 items by Osiris-gdw (Search limited from 18-November 18)


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#10381 Probelms with Cre-Recomination

Posted by Osiris-gdw on 27 January 2005 - 01:46 AM in Molecular Biology

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. :)



#9864 hep-2 & Hep-G2 cell line

Posted by Osiris-gdw on 10 January 2005 - 06:22 AM in Cell Biology

if someone could help me clear the difference between hep-2 & hep-G2 cell line, do both of these are hepatoma cell lines?
  i have been trying to grow hep-g2 cells with 10% FBS in DMEM but cells do not stick & dont divide i have been waiting for almost a week but there seems no response. i m using 25mm sq flask, even if few cells stick it seems as if they will take years to be cofluent. plz help me to solve this problem, thanks in anticipation, bye

Hi!
HEp-2 is a cell line derived from HeLa (they were are contamination in an originally hepatic cell line --> see ATCC for details) and so are a cervix carcinoma cell line, most useful, because they have a rather big nucleus.
HEP-G2 are hepatic cells.

So, if you need liver cells, HEp-2 are a bad choice!

If you need attached HEP-G2, just use poly-L-lysine to coat your dishes, that works quite well!

I hope, this helped...

Best wishes, Gerhard




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