Blunt end ligation problem - (Dec/03/2008 )

Here is my problem guys,

I am trying a ligation, one blunt end and one sticky end. I cut my pSec vector with Nhe I and EcoRV to produce a 7.8 Kb ligation vector. Also I cut another plasmid with the same two restriction enzymes to produce the insert which is 1.4 Kb. I gel purified all these pieces. My ligation reaction mixture is as follows.

Vector (25 ng)
Insert (13 ng) to give 1:3 Vector : Insert ratio
d water
Quick ligase buffer 5 uL
Quick ligase 0.5 uL
Total reaction volume = 10 uL

I tried this 16 hrs at 16 C, 5 min at 25 C
Also 2 hrs at 25 C
I tried 1:10 Vector:Insert ratio again at 16 C

Still I do not see any colonies after transformation. I did not dephosphorylate the vector, anyway I don't think the vector religates because I don't see any colonies on my plates. the positive control grows as expected.

Suggestions regarding reaction conditions, concentrations, vector, insert ratio, or etc..etc..are appreciated.

Thanks

check to see if the ligase is working. Is anybody else in the lab suffering from ligation problems?

Also once the ligation is done, and just prior to doing the transformation, run part of the ligated DNA onto a gel to see if the ligation reaction has occurred. If it has occurred you will see high molecular bands. (as a control, you can run some of a bit of ligation reaction mix which has not been exposed to T4 DNA ligase)

Is there enough bp between the NheI and EcoRV site to allow cleave of the restriction sites?

Check the DNA for salt contamination . Absorbance at 230nm and give a EtOH wash if it's high.

Prolong incubation to overnight if desired.

I think you have your ratios reversed. You should have quite a bit more insert than vector in your ligation, 2:1 or 3:1 insert to vector.
Let us know if it works.