cloning a fragment using RE sites - protocol given really fuzzy. (Mar/15/2006 )

Hello,

I've been given a project started by someone else, and i've got their protocol which makes nosense (either because i'm tired, or it's just silly).

Essentially i'm making a construct for pronuclear injection to generate a transgenic mouse.
I have plasmids for the vector, the promoter and the cDNA i want to use. Apparently there are maps, but they are so scant on information, that its scary.

The plan was to cut out the promoter, and clone in into the vector we want to use. Ok, done that.
check orientation (doing that now).
Next step is to cut the cDNA out of another vector (easy enough) using XhoI. Ok.
now this is where i'm confused. it has to be cloned into my vector (the one that has the promoter) in a SalI site.
How do you insert a XhoI fragment into a SalI site?
Does this mean i have to design primers with the SalI site in them?
Is there something else that is done here?

Any help here is greatly appreciated.

Vetticus

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I've got no idea, that won't fit.
I would indeed design primers with the SalI sites in it. But then you don't have to first isolate your fragment with XhoI, just do the pcr on the other vector.
But it has to be cloned behind a promotor, is there still another site? Do you have to clone between a SalI and an other site? Or is the orientation not important here?

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hi
xho and sal I are isoschizomers. So it's possible as the annealing region corresponds.

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You're right, I did look it up, but I'm afraid I looked at the wrong line for XhoI and picked XhoII (I've got a note before me with RGATCY written on it )

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Thankyou so much.
You're all brilliant.

Vetticus

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