New to Topo cloning - (Jan/12/2009 )

I'm new to Topo cloning and I have a question. It probably has an obvious answer, but it is eluding me!

I have an insert in the pCR4-TOPO vector, but it is in the wrong orientation! What do I use to cut it out again so that I can re-ligate?

Thanks, I'm a big Newbie!

-biogradstudent-

QUOTE (biogradstudent @ Jan 12 2009, 05:09 PM)

What do you mean by wrong orientation? My udnerstanding is that with the TOPO TA cloning systems it doesnt matter what orientation your insert is, as you just want to use it for sequencing. They are not expression vectors, therefore the orientation of your insert should not affect the outcome, as all the other elements in the vector will still be in frame and express accordingly (apart from the LacZ operon of course).

If you need a certain orientation (for a reason that I cant think of), just screen a few colonies as I doubt they'll all have the insert the same way.

hope this helps.

-almost a doctor-

I actually have 2 inserts in there and they need to both be in the same direction. Right now they are in opposite directions! One insert is in with a NotI site, the other using the Topo site. It's the Topo insert that I need to flip.

Thanks

-biogradstudent-

QUOTE (biogradstudent @ Jan 12 2009, 10:40 AM)

Either you make it again, or cut the insert in TOPO site, use the TOPO MCS for re-cloning the insert. It is a matter of looking up what enzymes you have available. If you don't find any, simply blunt end the both (vector and insert), and screen for the ones with right orientation.

Basically, it becomes a general cloning work, rather than TOPO-TA cloning.

-cellcounter-

Thanks for the responses! I appreciate the help. What I need to flip is conveniently flanked by EcoRI sites!

-biogradstudent-