Ligation woes - ligation? what's wrong (Sep/14/2005 )

Hi there,

I am trying to ligate 107bp into a 6.4kb vector. both of them are digested with EcoR1 and gel eluted by Qiagen column.I am using T4DNA ligase from Roche (Reaction conditions 25 degrees for 4 hrs)
when I do transformation and see the plates next day ,nothing is there. And I do include a positive control( pure plasmid).
could you suggest what went wrong?

Today I am preparing fresh competent cells and will transform the cells immediately.Do you think there will be a better chance this way?

For ligation ,what do you think are the critical parameters?

thanks
mili

If anything, I'd expect you to have lots of colonies without insert using this protocol (single cut means the vector has complementary ends that can ligate unless you've used a phosphatase). The fact that you're seeing no colonies leads me to believe your reaction is contaminated. Have you checked the ligation products on a gel after the 4 hour incubation?

Since your positive control is working, transformation efficiency is a possible culprit, but not altogether likely. Purified plasmid does transform easier (it's supercoiled, whereas the ligation product is not). If your ligation products look ok, new cells or ultacompetent cells could help. What was the transformation efficiency of your cells (ie, how many colony forming units per ng of DNA)?

The critical parameters for ligation, in my opinion (and in no particular order)...
- Plasmid size: too large means it won't get through the pores of the cell during the transformation, but your construct should not be a problem
- Insert:vector ratio: you want 3:1 molar ratio, approximately - not as important if you treat the vector with phosphatase before the ligation
- Purity of reagents: tiny amounts of contaminating nucleases will really mess things up
- Transformation efficiency vs. amount of product: obviously, if your cells are low efficiency, you need to start with more product, or make better cells.