Protocol Question

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Protocol Question

Post by dor940 » Sun Dec 06, 2009 11:06 pm

Okay so, I've been working on this problem for a while and still haven't figured it out yet. Our teacher gave us some hints but I am still not getting it. Any help would be appreciated.

You ligated a 1-kbp BamHI cut cDNA (lower case letters) with a 5-kbp BglII cut plasmid (upper case letters). This was fine for making your 300-residue protein in E. coli. But you want to make the protein in mouse cells also. To do that you need to reclone the same cDNA into a mammalian expression vector, which has unique BlgII and BamHI sites. How would you do it? Show work.

1) You must write out the restriction sites for all enzymes that you need to use for first cloning and the second cloning.
2) Pay attention to what happens to the restriction sites after the first cloning.
3) Think before taking any step for the second cloning. PAY ATTENTION TO THE SEQUENCE OF RESTRICTION SITES AT ALL LEVELS.
4) Use the NEB website to learn about the relevant sites and enzymes.
5) Then proceed with cloning into the mammalian expression plasmid.
6) Need to know restriction sites and restriction enzymes
7)Need to write down sequence and cut sites and show them

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Post by JackBean » Fri Dec 11, 2009 8:07 am

There is nothing easier than
or if you really need to use NEB site, just look for
And the same for BglII ;)
On NEB web site you have sequences, where these enzymes cut and in what place they actually cut.
Can you imagine, what happens, if you put together one piece cut by BamHI and other piece cut by BglII?

Cis or trans? That's what matters.

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