Cutting the ends of dna

Frederik Boernke boernke at nospam.ipk-gatersleben.de
Fri Oct 29 15:52:27 EST 1999


Aidan Weatherill wrote:
> 
> I am building a construct by pcr'ing up 3 fragments, that I want to join
> together. the pcr oligos contain restriction sites at the ends so I can cut
> the products and ligate them in a sticky end way (and guarantee
> orientation).
> 
> I am recovering the correct ligations by pcr'ing up from the ligation with
> the most external oligos.
> 
> I have had no luck in recovering the correct construct. I believe that they
> are not sticking because the enzymes are not cutting at the ends. the
> enzymes are sac1 bamh1.
> 
> If someone can suggest ways of increasing the efficiency of a) cutting at
> the ends of molecules and b) doing triple ligations, I would be very (very)
> happy
> 
> cheers
> 
> Aidan
> 
> aweather at molbiol.ox.ac.uk

Hi Adrian,

I would suggest to clone the three fragments into a pcr vector first
than reisolate and ligate them into the target vector one after the
other. Another thing would be ligation pcr where you amplify your 
fragments in seperate reactions with your primers having complementary
sequences to the neighbouring fragments thus after mixing the fragments
and doing pcr with 5' and 3' primers of the entire fragment you can
recover a "ligated" fragment. 
It's a bit hard for me to explain but I hope it helps.

Ricky

*******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de



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