Ligation of pBBR with PCR fragments doesn't work - (Jun/12/2007 )
Hi to everyone,
I have a probably quite common problem... I'm trying to ligate a double-cut (Hind III, Xba I) vectorplasmid pBBR (MCS5, 4,8kb) with PCR fragments that were digested by Hind III and Xba I. Unfortunately I don't get any clones after transformation with DH5 alpha.
I'm already performing the ligation over night at 15°C and restricted my vector in a two-step restriction (first Xba, then Hind) with a purification (QIAGEN) after every step. And I performed a pDrive-cloning (TA) with my PCR products to extract correctly cut Insert from a 2% agarose gel... but still I don't get any clones after transformation, although the competent cells should be OK (I didn't check the competence, but they worked fine with another transformation).
Does anyone use pBBR as a vector-plasmid and had the same problems... or any more clever suggestions regarding a ligation? 
Thanks for your help...
what ratios and amounts of insert and vector are you using in ligation. How long do you digest the vector and PCR product? have you run a gel with both insert and vector to check concentration and integrity on gel.
Any possibility of ligase or buffer getting contaminated?
Any possibility of ligase or buffer getting contaminated?
I used a ratio of 1:3 vector to insert but checked 1:1 and 3:1 aswell... I usually just compared the amount of vector to the inserts on a 1% agarose gel
Restriction was performed for two hours with 2ul (40units)enzyme in 100ul and then another 1ul (20units) was added for restriction over night at 37°C
Yes, I checked both, vector and insert on agarose gels (see above)
I suspect that you are overdigesting your DNA and damaging the ends. Most enzymes have some low level exonuclease contamination that becomes evident when too much enzyme or too much time is used. How many ug of DNA are in your restriction digest? NEB recommends 0.13 Units/ug of DNA for overnight cuts, while you have 60 Units in your overnight.
I also don't understand why you are using TA cloning for restricted DNA fragments.