Question

The average molecular weight of a nucleotide (base + deoxyribose + 1 phosphate group) in DNA is 308g/mol of 308 Daltons (Da). This number is the average molecular weight of
A = 312.2 g/mol
G = 328.2 g/mol
C = 288.2 g/mol T = 303.2 g/mol
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R-plasmid was used for the transformation experiment

a.What is the size of this plasmid (in kilo base pairs; kbp)? How many bases is this?
b. What is the molecular weight of one plasmid molecule?
c. You used 25μl of 1ng/μl plasmid DNA per transformation. How many grams of plasmid DNA
did you use? How many moles of plasmid DNA is that? How many molecules of plasmid DNA
is that?
d. Assuming each transformed cell pick up one plasmid molecules during the transformation
process, what percentage of DNA molecules successfully entered and replicated inside the
competent cells?

Answer 1

a. R-plasmid or Resistance plasmid ranges from 80 to 95 Kbp in size. 1 Kbp = 1000 bp; so in terms of bases, R-plasmid would be between 80000 to 95000 base pairs.

b. Given the average molecular weight of a nucleotide base = 308 Daltons.

the molecular weight of R-plasmid can be calculated, assuming a size of 80000 base pairs:

Total number of nucleotides = 80000 * 2 = 160000

Molecular weight = 160000 * 308 = 492,80,000 Daltons = 4.928 * 10⁷ Daltons.

Therefore, the molecular weight of one molecule of R plasmid, (assuming its size as 80000 bp), would be 4.928 * 10⁷ Daltons.

c. given, plasmid DNA used for transformation = 25 uL of 1 ng / uL

total amount of plasmid DNA used = 1 * 25 = 25 ng

1 ng = 10^-9 g, therefore the amount of plasmid in terms of grams = 25 * 10^-9 g or 2.5 * 10^-10 g.

Molecular weight of the R-plasmid = 80000 g / mol. or 80000 ng / nmol.

Amount of plasmid used for transformation = 25 ng.

The number of moles of plasmid used = 25 / 80000 = 0.00031 nmol or 0.31 pmol.

The number of molecules in one mole of any substance is given by the Avogadro number: 6.023 * 10²³ molecules.

1 pmol = 10^-12 mol.

Number of molecules per pmol = 6.023 * 10²³ * 10^-12 = 6.023 * 10¹¹.

Therefore, the number of molecules in 0.31 pmol = 0.31 * 6.023 * 10¹¹ = 1.9 * 10¹¹ molecules.

The percentage of molecules that entered inside competent cells depends upon the number of competent cells present in the tube.

A good preparation of competent cells should yield 10⁸ colonies per ug DNA. In this case, we have used 25 ng DNA, so the number of colonies would be: 0.025 * 10⁸ = 2.5 * 10⁶ colonies. Which would mean that many cells have taken up one molecule each of the plasmid. So the percentage of DNA molecules that entered the cells successfully here would be:

2.5 * 10⁶ / 1.9 * 10¹¹ * 100 = 1.3 * 10^-5 * 100 = 1.3 *10^-3 = 0.0013%