Blunt end ligation problem - (Dec/02/2008 )
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
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
I think increasing your vector amount to 150ng (and increasing insert amount accordingly) will help. 25 ng of vector at 8kb size is too low in my view (and experience).
I'd also increase the amount of DNA.
Are you sure your ligase is okay? It might be worth checking it... for example, linearise and gel purify a vector, then compare the number of colonies in ligase -ve and ligase +ve reactions.
Ginger