Calculating transformation efficiency? - (Nov/26/2008 )

Hey guys,

Can anyone help - I need to calculate transformation efficiency: no of colonies / ng of dna plated

6ul vector plus pcr product added to 45ul ligation mix
Ligation mix added to 100ul competent cells and then 1000ul SOC medium
50ul plated
(my initial dna concentration is 0.15ug/ul)

If someone can help with taking into account these dilutions and plugging them into the equation that would be great, im awful at maths

Thanks!!
Jennycharlie

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Transformation efficiency is usually applied to competent cells using control plasmid rather than the recombinant vector of your experiment, and it's usually expressed in terms of colonies per ug DNA.

You need to know how many ng or pg vector you have throughout the experiment, so factor this into your dilution calculations: how many ng vector do you use in the ligation reaction (i.e., concentration); how many ul of the ligation mix do you add to the cells (what volume of cells?; dilution factor); how much SOC do you add in the recovery stages(dilution factor); and how many ul do you plate out (it's often useful to plate out a number of volumes to give some flexibility to your calculations; dilution factor)?

From the numbers you give, here's my 5 cents worth:
6ul@0.15 ug/ul=0.9ug vector.
In 51 ul ligation mix, plus 100ul cells and 1000 ul SOC: 0.9 ug in (51+100 + 1000) = 0.0007819 ug/ul (=0.7819 ng/ul)
Therefore you plated 39 ng vector in 50 ul.

The big assumptions include how much of the vector is cut (if it isn't 100.0%, your efficiency is going to reflect non-recombinant molecules) as well as how much of the digested vector ligates without insert and how much incorporates your insert. You also assume all of the vector is taken up. For these reasons, I've not heard of people bothering with the experimental samples, but only the control, to see how competent the cells are.

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