ligation problem - (Aug/21/2007 )
i have been cloning 600bp into pBIN121 vector.. both vector and insert are digested with Hind III and Xba I..i tried 1:3 conc and i didnt get any colonies.. but competent cells and ligation solutions are fine... i cant trouble shoot what is problem...
first of all let me ask is it 1:3 in favor of the vector or the insert? I hope it's the insert. And is this the molar ratio or mass (or something else)?
Do you have any antibiotic resistance marker on this vector (I saw somewhere that it should be rifampicin)? Did you use the right amount?
That's my 2 cents. Maybe if you included some more information...
You can test the digestion and the ligase by some quick ligations. First, add ligase to a small aliquot of the insert and to some of the DNA ladder, then run a gel. You should get high MW bands. If you only get a dimer-sized insert band, one of the REs has failed to cut well. If the DNA ladder doesn't make large DNA fragments, there's something wrong with it.
How did you purify the digested insert? Gel extraction is good, but you can have some pretty big losses. Ammonium acetate/EtOH precipitation is quite good, especially as the termini of the insert don't precipitate well.
thanks.. i used kanamycin as antibiotic resistance marker... for 100ml, 100 microlitre is added.. then ligation temperature is 16 C for overnight and vector :insert ratio is 1:3.. i used 1ul of vector and 3ul of insert... i got 2 colonies but i cant screen it out... when i cut with HIndiii and xbai , i found shift but insert didnt release... also in colony pcr using gene specific primer, i got bands even in neg tive control that is pBIN121 vector... i cant confirm it... what can i do...
The 1:3 ratio refers to the molar ratio of vector to insert, not the volume. This article on openwetware describes how to calculate your ligation mix properly.
I recently had a similar problem, are there any GATC sequences around the new(vector plus insert) Xba I site? I cloned a fragment using double Xba I, I got colonies but couldn't release during screening (I could linearise), however sequencing (and alternative RE digest) confirmed that the the fragment was indeed there. Xba I is dam methylase sensitive (GATC), traditionally TCTAGATC is a problem, in my case, surprisingly it was GATCTAGA on one of the two sites that was causing the digestion problem.