Technique / Molecular Biology / Cloning library construction and screening


help needed with three fragment ligation



help needed with three fragment ligation

I have a puzzle with regard to a ligation I am trying to perform. I want to ligate three pieces of DNA together, one of 7.1kb, one of 3.6kb
and one of 1.9kb, to form my complete plasmid for bacterial transformation.All the fragments use different sticky ends to minimise problems with the pieces religating in the wrong orientation/order but it doesn't seem to help. I have done the reaction a number of times without success. I would be eternally grateful to anyone out there who has any bright ideas/tricks of the trade that you have used for ligating multiple fragments of DNA. ... ...

Last update 22-Nov-2001, Rating of 0 votes.

Post your message in Cloning library construction and screening forum

Write your comment

By S.Sathish on 20-May-2003
These fragments that you had mentioned are these PCR amplified and does it have the restriction site flanking the primer sequence, and what are the restriction sites.I think 7.1 kb is the plasmid 3.6 and 1.9 are inserts, what was the ratio of the insert : vector concentration u used for ligation.
Rating:

Reply
By FLH on 22-Nov-2001
use EcoRI/XbaI for the two sites of the vectors,chose NotI for the other site of the frgment.Someone finds it works.
Rating:

Reply


Please Login or Register to Post