RF cloning, have anyone tried it?

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oppox
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RF cloning, have anyone tried it?

Post by oppox » Thu Jun 29, 2006 9:21 pm

At the moment im trying to do a RF cloning. RF stands for restriction free. What u do is that you use primers about 50 bases long, half of them is complementary to your insert of interest and hal to the vector you want to clone your insert into.

You amplify your gene with the primers and then using the gene with added primers as one big primer and the vector as template in a quickchange reaction. Then treat it with dpn1 and voila you have a newly syntesized vector with your insert. The thing is I dont get any band on the gel (agarose) in the region of the right size, vector + insert and im running the quickchange reaction for 35 cycles. I start with 1 microliter vector (20 ng) in 50 microliter quickchange reaction.

have anyone tried this method before? and experienced any problems?
I think its a very neat and cool reaction in theory but it seems harder to do in reality. Thx in advance.

Mihtrio
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Post by Mihtrio » Tue Jul 18, 2006 9:28 pm

yes, i think so... :)
Mihtrio is here

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