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GFP fusion protein

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GFP fusion protein

Postby biology_06er » Sat Nov 19, 2011 10:49 am

Hi there,

So I'm currently in the process of ligating my protein of interest with a Pet vector containing GFP. Once I've done that, I plan on transforming into BL21 cells, and then hopefully I will have some positive colonies..to check for +ve colonies I plan on carrying out single colony PCR...as thats what I've done in other situations...buut if I add some IPTG to my LB-agar, can I view the plate under UV light and is it possible some colonies will fluorese green and then I can just grow that colony overnight and skip the single colony pcr step.

Cheers,
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Postby daniel.kurz » Mon Nov 21, 2011 2:33 am

The viewing is one way that you can check that you've done your ligation successfully.
Its the way that I would do it. I would suggest that in either case you do a mini-prep (Promega makes these and many other companies) and send DNA for sequencing. Make sure that you don't autoclave your LB with ITPG in it because it can break down under high temp. Add it like you would antibiotic as the LB cools before pouring it out.

In either method, the problem is that you don't know if your protein is in correctly. Are you using blunt end or sticky end restriction enzymes to make the cut between GFP and protein? If you are using blunt end, you could get the protein in backwards and then the whole GFP+protein fragment goes in right so the GFP gets expressed by some odd protein gets made. Thats why I suggest that you send for sequencing because you can check the positioning and also no random mutations were put in.
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