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ligation probs


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2 replies to this topic

#1 sagar

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Posted 06 April 2009 - 09:01 AM

hi all

i have certain queries about the general approach for the ligation procedures .i have been trying to clone a 1.4kb insert into a 2.8kb vector but each time i get vector-vector ligaion but not vector-insert ligation.ihave been setting up the ligation reaction as follows

vector:100-150ng
insert:as per calculatiion from 3:1 insert:vector molar ratio
T4 Dna ligase:1 microlt
buffer:1 microl
total volume :10 microlt

i thought the vector concentration is very high thus i was getting concatamer thus i reduced the concentration of vector to 50ng.the reactions being

vector:50ng
insert:as per calculatiion from 3:1 insert:vector molar ratio:75ng
T4 Dna ligase:1 microlt
buffer:1 microl
total volume :10 microlt

mostly its recommended that the vector concentration should be 50ng but i didnot get any colonies.after searching for other methods i found that the concentration of both vector and insert together being in the range of1- 10ng/ul cause efficient ligation

molecular cloning experts can any one plz help

#2 phage434

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Posted 06 April 2009 - 11:01 AM

Tell us what the vector and insert DNA are, where they came from, and how they were prepared. Usually this is the problem. Have you tested the efficiency of your competent cells? How are you doing the ligation? How are you transforming? Tell all.

#3 olfaction

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Posted 11 April 2009 - 01:33 PM

I do not know anything about the vector you have used but If you are trying to ligate it into restriction enzyme cut sites why do not you CIP the hell out of it?




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