the 15micro liter of competent E.coli 10^9 cfu/ml were transformed using 0.5ng of a 5kb plasmid DNA to which 950 micro liter of SOC medium was added only 50 micro liter of these plated on selective agar plate. after and 12 hrs incubation at 37°C 90 colonies were observed. calculate the efficiency of the transformation in cfu/microgram of DNA. calculate tge percentage of transformed cells.
Answers
Explanation:
1-For transformation, you could use very less DNA, 1 ng. You need to run a positive transformation control and calculate your transformation efficiency.
2-Information: You must use plasmid for calculating transformation efficiency. If you use a ligation product, your calculation will be incorrect due to the low amount of circularized DNA in a ligation reaction.
3-Calculate your transformation efficiency with the following equation (CFU is colony forming units): (# colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA
The measurement "ng of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:
volume of plasmid used in µL x concentration of DNA in ng/µ x (volume plated / total reaction volume)
Example: You transformed 1µL of 0.05 ng/uL plasmid from the Transformation Efficiency Kit (note: 50 pg/µL = 0.05 ng/µL) into 100 µL of competent cells. You added 900 µL SOC to your cells to get a total reaction volume of 1000 µL and then plated 100 µLs of the transformation. The plate has 250 colonies on it the next day.
ng of DNA plated calculation: 1 µL x 0.05 ng/µL x (100 µL plated / 1000 µL total reaction volume) = 0.005 ng DNA plated
Efficiency calculation: (250 colonies / 0.005 ng) X 1000 ng/µg = 5 x 107 CFU/µg DNA