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GFP

Postby biology_06er » Sun Oct 23, 2011 10:44 am

Hi there,

So I recently ligated my gene into a Pet32a vector using restriction sites using EcoR1 and Xho1. From another Pet vector I have to create primers for the GFP insert containing BgII and EcoR1 sites. Just wondering, once I amplify my GFP (using BgII/EcoR1 sites I assume), how do I ligate it into my original construct..Do I cut my original constuct with EcoR1 and BgII (because looking at the vector map, Pet32a has a BgII site and ligate?)

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Postby JackBean » Sun Oct 23, 2011 11:00 am

you should thing about this before you start your work, because you should be sure, that e.g. BgII won't cut in your gene of interest or that it doesn't cut your vector more times. But yes, I would expect that.
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Re:

Postby daniel.kurz » Mon Oct 24, 2011 2:49 am

JackBean wrote:you should thing about this before you start your work, because you should be sure, that e.g. BgII won't cut in your gene of interest or that it doesn't cut your vector more times. But yes, I would expect that.

I agree with Jack Bean. If you're not careful, you will end up with your gene of interest in the wrong spot. My experience is that so long that your cut doesn't degrade the end and become blunt ended and doesn't get a strange mutation it should ligate in nicely. Best of luck.
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Re: GFP

Postby biology_06er » Mon Oct 24, 2011 9:48 am

Hi there,

Thanks for the replies...I had a relook and it seems my GFP insert already has BgII and EcroR1 inserted (as it would as it is already in the Pet vector) then i wouldnt need primers right?... I could cut the GFP insert straight from the Pet vector using BgII and EcoR1 and likewise cut my original construct with those 2 restriction enzymes and ligate?
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Postby JackBean » Mon Oct 24, 2011 10:07 am

wait a moment, so you have a gene, which you want to clone into pET vector and GFP, which you have in a pET vector? So why do you want to cut the GFP?
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Re: GFP

Postby biology_06er » Mon Oct 24, 2011 11:13 am

Hi,

I have 2 pet vectors...one containing GFP and one containing my insert.....the GFP was has a bgii and ecor1 site and my insert has a xho1/ecor1 site....the plan is to fuse my insert with GFP so im thinking if the gfp is already in a pet vector, i need to cut it out and insert it into the vector contiaing my insert?
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Postby JackBean » Tue Oct 25, 2011 12:02 pm

OK, I see. Then you should be able to just cut the GFP out of the vector, but remember, that with PCR you increase the number of copies, with restriction you will get at best the same number of GFP copies as you have vectors.
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Postby daniel.kurz » Fri Oct 28, 2011 5:57 am

Agreed.
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