TP33\TP33
Calculations with DNA
Following the ligation of a DNA fragment to a plasmid vector, the recombinant molecule should be introduced into a host bacteria in order to replicate.
The efficiency of transformation is a quantitative measure of how many cells take up a plasmid. It is represented in terms of transformants per gram of plasmid DNA.
The computation is shown in the following problem:
A 25 µL ligation reaction contains 0.5 µg of DNA of an AmpR (ampicillin resistance) plasmid cloning vector. 2.5 µL of the ligation reaction is diluted into sterile water to a total volume of 100 µL. 10 µL of the dilution are added to 200 µL of competent cells (cells prepared for transformation). The transformation mixture is heat shocked to increase plasmid uptake, and then 1300 µL of growth medium are added. 20 µL are then spread onto a plate containing ampicillin and the plate is incubated at 37°C. The next day, 220 colonies appear on the plate. What is the transformation efficiency in terms of transformants pre µg of vector DNA?
Exact M.W. of ssDNA (e.g., Oligonucleotides):
M.W. = (A x 313.2) + (T x 304.2) + (C x 289.2) + (G x 329.2) + 79.0ª
Competent cells prepared for transformation can be purchased from commercial sources and may exhibit transformation efficiencies greater than 109 transformants/µg DNA. This is usually measured using the pUC19 vector. If we assume that one transformant has taken up exactly one plasmid, what is the percentage of plasmids that end up being transformed into cells?
Calculations_for_Molecular_Biology_and_Biotechnology:_A_Guide_to_Mathematics_in_the_Laboratory_2_ed
https://www.sciencebuddies.org/science-fair-projects/references/genomics-g-c-content-calculator
https://bitesizebio.com/11120/a-guide-for-solving-your-lab-math-problems/
https://bitesizebio.com/20669/how-to-calculate-the-number-of-molecules-in-any-piece-of-dna/
http://www.uvm.edu/~btessman/calc/
http://www.uvm.edu/~btessman/calc/calc2.html
http://www.uvm.edu/~btessman/calc/calc2a.html
https://openwetware.org/wiki/BISC220/S10:\_Sample\_Calculations\_Lab\_2
Literature and Sources
Stephenson, Frank Harold. 2010. *Calculations for Molecular Biology and Biotechnology: A Guide to Mathematics in the Laboratory*. 2nd ed. Academic Press/Elsevier.