Question

Background Calculating Transformation Efficiency Restriction digest & ligation: You add restriction enzymes to 0.05ug of each plasmid in separate pAMP and pKAN tubes tubes and heat the restriction enzymes by placing them in a hot water bath for 20 minutes. Afterwards, you label 1 tube pAMP/pKAN and combine 4 ul of digested pAMP, 4 ul of digested pKAN, 1 ul of ligase, and 1 ul of ligase buffer and leave it to incubate overnight. You then label an empty tube -pKAN/-pAMP which will be used as your control (no plasmids). Bacterial Transformation: To the 2 microcentrifuge tubes labeled +pKAN/+pAMP and -pKAN/-pAMP you add 250 µl of transformation solution (0.05M CaCl₂). Using aseptic techniques you transfer bacteria colonies into the -pKAN/-pAMP and +pKAN/+pAMP tubes making sure to avoid cross contamination. You incubate the tubes for 10 minutes in ice, transfer them to a water bath for 50 seconds, and then back in ice for 2 minutes. You then pipette 250 uL broth into each tube and incubate them for 10 minutes to make the bacteria happy again. Afterwards, you pipette 100 ul of each transformation mixture and spread it onto the appropriately labeled agar plates. You parafilm and incubate the plates of E. coli overnight. The next day you got the following results.   Plate Label # of colonies - LB agar Too many to count (TMTC) - LB pAMP/pKAN No growth + LB agar TMTC +LB pAMP/pKAN 2 The questions that I need help on: Calculate the transformation efficiency in CFU/ug of DNA from the +LB/AMP/KAN plate. The concentration of the pKAN/pAMP is 0.1 ug/ul. The amount of plasmid DNA spread on the plate was 1 ug. Determine the number of colonies growing on the LB/amp/kan agar plate based on the information provided. Determine the amount of plasmid DNA (in ug) spread on the LB/amp/kan agar plate from the information provided. DNA spread (ug)= Volume spread on plate (ul) X DNA in transformation (0.05 ug)                                            Total volume of transformation                                        (add up all the highlighted volumes) Calculate the transformation efficiency (CFU/ug) by dividing up the number of colonies on the plate by the amount of DNA spread on the plate. Basically divide the answer from #1 by your answer to #2

Answer 1

Given information:

After restriction digestion and ligation,

+pKAN/+pAMP Labelled tube	-pKAN/-pAMP labelled tube
4µl digested Pamp+ 4µl digested PKAN+1 µl ligase+ 1 µl ligase buffer	No plasmid
250 µl CaCl2	250 µl CaCl2
Bacterial colonies	Bacterial colonies
250 µl broth	250 µl broth

100 µl of each transformation mixture was spread on the following labelled plates and the CFU obtained are also enlisted below:

Plate Label	# of colonies
- LB agar	Too many to count (TMTC)
- LB pAMP/pKAN	No growth
+ LB agar	TMTC
+LB pAMP/pKAN	2

Concentration of the pKAN/pAMP = 0.1 ug/ul.

Amount of plasmid DNA spread on the plate = 1 ug.

Based on the information provided,

number of colonies growing on the LB/amp/kan agar plate = 2CFU/100 µl or 0.2 CFU/1000 µl or 0.2CFU/ml

Amount of plasmid DNA (in µg) spread on the LB/amp/kan agar plate:

DNA spread (ug)       =       (Volume spread on plate (µl) X DNA in transformation) /

                                           Total volume of transformation

Here, Volume spread on plate= 100µl

            Dna in transformation= 0.05µg

Total volume of transformation= 500µl (approximately)

Putting these values in the above formula,

DNA spread (µg)= (100µl X 0.05µg)/500µl

                                       = 5/500

                                    =0.01

Amount of plasmid DNA spread on the plate= 0.01µg.

Transformation efficiency       = No. of colonies on     plate/ Amount     of DNA spread

=2/0.01

=200CFU/µg