Question

You had a total of 1.0 mL of "transformation mixture" including the cells and DNA. How many TOTAL transformants would you have seen if you had plated the whole 1 mL of transformed cells? (There are many ways to approach this questio, given your numbers above) . Please explain you reasoning.

Below are the number of transformants I counted for each plate. There are two plates of each 25, 50 and 100 microliter.

25 microliters plated: 249, 186

50 microliters plated: 222, 81

100 microliters plated: 440, 496

1000 microliters of sterile TE uffer and 1 microliters of purified plasmid DNA was added into a 12 mL tube.

50 microliters of competent cells were added to the chilled tube

5 microliters of plasmid DNA were added

The solution was chilled for 20 minutes. The solution was then heat shocked into a 42 degree water bath for 45 seonds, then chilled for 2 minutes

1 ml of preheated "SOC" liquid media was added to the solution

Answer 1

1.Dilution of the ligation mixture with T.E buffer is important for transformation efficiency as the components of ligation mixture can inhibit transformation.

2. As we see from the above example the number of transformants do not increase with increase in the volume that is plated ( from 25ul to 50 ul).

3. However given the amount of DNA (ug) used and number of transformants obtained , one can calculate the tranformation effiency of the competent cells

Number of transformants/?g of DNA

X

final vol at recovery (ml)/vol plated (ml)

=

Number of transformants per ?g