hi,
im getting no colonies or colonies with no insert.
I think it is probably ligation issues. the insert are self ligating but no vector insert product..how can I fix it..no problem with transformation..i cut an insert out of the vector..so no problem with digestion.
from right to left (ladder, vector, ligation 1 to 8)..vector 6100bp (top band), insert 2000 to 1800, right to left bottom band), insert self ligation (middle band)..help..im doing it so many times..so frustrating.
Hi you should calculate vector to insert ratio for me 1:3 ratio works best. you can try this link http://www.insilico.uni-duesseldorf.de/Lig_Input.html
How long do you incubate ligation reaction and at what temp.??
hi gopal, thanks for reply..i incubated overnight at 16..i used 1:3 ratio..also do u know how many bands im supposed to get in the ligation reaction? i use the same condition with other proteins and was very successful every case..this is the only protein giving me hard time..don't know whats wrong.
I usually never run ligated product on gel.. I directly tranform it and screen colonies later. Do you deactivate restriction enzyme after digestion before ligation? also I purifiy digested product so remove any excess salts from digestion buffer etc. after purification I elute in small volume of dh20 like 5ul or so, and use it for ligation.
As there may be possibility that soem of insert and template might not have liagted and they will appear as multiple band.
Hi Fazlur,
Do you do a restriction digest to confirm the success of your ligation? I am also a bit surprised by the intensity of the bands you get on the gel from the ligation. How much DNA do you use to perform the ligation?
Best Simone
Hi, simone..i use approximately 100ng in 10ul reaction. for ligation..how can I confirm ligation by restriction digest? It seems clear that the ligation did not work since I don't see any band above the vector alone reaction band..
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