about ligation problem - insert and vector size (Feb/10/2007 )

hi all
i am stuck again with ligation problem

now i want to ligate a vector of 6.5 kb with a insert of 7.2 kb

in ligation i used 1:1 ratio

do u have any suggesstion please give me

I would suggest using ratio of 1:5 or even higher. Also use more DNA, like instead of using 20ng of each use 50 or even 100ng of each. This may sound ridiculous, but for some resistant ligations, this works.

Also verify that the cells you are using are good.

Good Luck !!!

Try high concentration of ligase as well. i know there is some "powerful" ligase concnetrated 2000 times, which saved me from that bloody blunt-ended ligation. Might be perchursed from promega. i am not sure.

i am tryine blunt end ligation .
scolix about ur suggesting ratio.........
are u suggessting me to use 1 vector: 5 insert??? or the reverse???

my labmate told me to try the reverse

I think 1 vector: 5 insert will be better instead of reverse.
Oh.. but both insert and vector are about the same size. Perhaps you can try 2 vector: 1 insert first. And try overnight ligation.

As its a blunt ligation, hopefully you have dephosphorylated the vector.
For ligation use insert:vector 1:5 ratio
if u have enough DNA you could try the other way as well just to see how they work.

hi
i think you may try to increase the time of incubation for overnight.
i sometimes tried to keep at room temperature for 3 hours and then at 4 overnight.
try to increase your insert.
good luck

i incubate in 16c for overnight.
for blunt eng ligation is 4 c better than 16c???

try increasing the starting concentration of dna in a large volulme. we usually do 16o overnight. it works well for blunt end.

I leave all my ligations at RT.