Ligation problem - (Jul/24/2008 )

I am trying a ligation and it did not work so far. So I like to hear your suggestions to make it work. My vector is 4 Kb and the insert is 1.3 Kb. Both vector and insert have restriction sites Kas I and Nhe I. My positive control always grows after the transformation, no colonies in my negative control also my ligation products never grow. I tried quick ligase ( 5 min at room temperature) and T4 ligase (overnight at 4 C). For ligations I used 25 ng of vector and 24.4 ng insert following 1:3 molar ratio. For the quick ligation the total volume was 15 micro liters and for the other it was 10 micro liters. I tried different concentrations starting from the above mixtures like a dilution series.

I like to know what else I should try and where would be the problem.

Thanks

How long did you digest the vector and insert? Too long digestions can hurt sticky ends resulting in no ligation. I have no worked with KasI so am not sure how good the enzyme is? nhe I should be fine. I would digest not longer then 1-2 hrs and try ligation again. Also verify the DNA conc. by loading it on gel. Some spectrophotometer readings are not too reliable.

Good Luck !!!

Did you gel purify your fragments? Did you limit their UV exposure? Did you dephosphorylate your vector. A 'yes" could help your ligation.

Sometimes it is not ligation problem. It is transformation/medium/antibiotics... problems.

Are you sure your controls are believable?

QUOTE (kj2008 @ Jul 24 2008, 03:46 PM)

I am trying a ligation and it did not work so far. So I like to hear your suggestions to make it work. My vector is 4 Kb and the insert is 1.3 Kb. Both vector and insert have restriction sites Kas I and Nhe I. My positive control always grows after the transformation, no colonies in my negative control also my ligation products never grow. I tried quick ligase ( 5 min at room temperature) and T4 ligase (overnight at 4 C). For ligations I used 25 ng of vector and 24.4 ng insert following 1:3 molar ratio. For the quick ligation the total volume was 15 micro liters and for the other it was 10 micro liters. I tried different concentrations starting from the above mixtures like a dilution series.

I like to know what else I should try and where would be the problem.

Thanks

I'd try to ligate ON at 16°C, and tweak the ration of vector to insert a bit. What about 1:10 (as the insert is pretty big and hard to get into the vector).
Also, you could check the amount of vector you are starting with at the very beginning. It usually works better if you use less vector (smaller probability to get inclomplete digestion and so on...), and you could do the digest 4h.
Other than that I recommend you sacrifice some biscuits to the god of ligation

Those are the things that usually help in my clonings, I whish you good luck

dedee

Sometimes longer ligation period helps....mine works at 1-2 days time at 4C with T4 DNA Ligase. Good luck!

Did you test the ligase? Treat some DNA ladder for 15 minutes at RT, then run on a gel. Do the same with the insert. If you don't get a shift in the DNA ladder, and if the insert doesn't make a ladder, your ligase is off. If the shift in the ladder happens, but you get only dimers, or mostly dimers in the insert, one of your restriction enzymes is off.