GFP-positive water?

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lieffenno
lieffenno's picture
GFP-positive water?

I'm genotyping about 100 clones that i have transfected with a gfp reporter plasmid (co-electroporation with a rtta cassette that has a resistance gene). The gfp does not have a resistance cassette so I am typing all of my clones for gfp. The issue is that my negatives keep coming up positive. I have switched all of my reagents, tried two different primer sets and protocols, and am extremely cautious with my equiptment.

My negatives are 1) h20 and 2) genomic mouse dna that is gfp-negative. One of the protocols I have used includes an internal control which amplifies the mouse negative but not the water.

Any suggestions?

Thanks,
Lief Fenno

Fraser Moss
Fraser Moss's picture
Can you tell us prescisely

Can you tell us prescisely which plasmids the GFP and the rtta are on.

GFP plasmids usually come with Kayn Res.

What antibiotic are you selecting with for the rtta cassette?

lieffenno
lieffenno's picture
The GFP plasmid is built from

The GFP plasmid is built from a pUC119 backbone, which has inserted a large 5' chunk of rat TH promoter, an intron, eGFP, and a polyadenylation site. When i run this plasmid through the same typing protocol I get a band that is much brighter than any of the other bands, although this may be due to differences in DNA concentration. When I linearized the plasmid for electroporation the digestion excised the backbone, leaving only the TH/intron/eGFP/pA build.

the rtta is built off of pCAGGS and has a puro cassette.

Thanks :)

Fraser Moss
Fraser Moss's picture
lieffenno wrote:The GFP

lieffenno wrote:

The GFP plasmid is built from a pUC119 backbone, which has inserted a large 5' chunk of rat TH promoter, an intron, eGFP, and a polyadenylation site. When i run this plasmid through the same typing protocol I get a band that is much brighter than any of the other bands, although this may be due to differences in DNA concentration. When I linearized the plasmid for electroporation the digestion excised the backbone, leaving only the TH/intron/eGFP/pA build.

the rtta is built off of pCAGGS and has a puro cassette.

Thanks :)

I presume that when you linearize you purify the GFP fragment from the PUC19 backbone before you electroporate?