volume of ligation product - (Dec/26/2006 )

why and how does the volume of ligation product loaded into the competent cells matter? Can anyone resolve my doubts ? thanks !

I setup ligations in a final volume of 10 ul, when i was not getting my colonies when using 2 ul per 40 ul of competent cells i started using the whole 10 ul but its really a 5 fold difference in concentration of dna per transformation.. i dont think that should affect if you have good competent cells .. anyway.. i am sticking to loading the whole thing

Normally my ligation mixture is 20ul and I use 1-2 ul for most transformations but I would go upto 5ul sometimes on 50ul of cells.

But if u electroporating, people suggest to use less of ligation mixture, I am not sure y.

I understand that if you are electroporating cells, cells are prepared by several washes in just water, if the dna was resuspended in buffers that contain salts, by increasing the volume you will also decreased the transformation efficiency, not the case if you use dna in water. where the conductivity is better.
anyway... if my l00 ng in 10 ul if i use 2 ul would be like using 20 ng. should be enough but still i use the whole thing (considering that when transforming plasmid dna 80 ng/ul , 1 ul ins enough to have tons of colonies)

Chemically competent cells are in an osmotically fragile state, prepared with complex salt mixtures. Adding ligation mix in high volume changes the salt composition quickly and substantially, leading to cell death. They are also highly sensitive to any type of detergent, such as the triton X-100 found in some restrction digest buffers (EcoRI e.g.), even when present in the small amounts carried over from digest to ligation to transformation.

Electroporated cells are much more robust, and as mentioned above, the major concern is the conductivity of the cell + ligation mixture which cannot be so high that the cuvette arcs during transformation. Drop dialysis can be used to reduce salt concentration if this is a problem.

i think most protocols coming with the ligation mixes say not more than 1/10 of the ligation mix.