You measure the OD600 of your culture of chloramphenicol-treated XL1-Blue/pTrc99
ID: 146334 • Letter: Y
Question
You measure the OD600 of your culture of chloramphenicol-treated XL1-Blue/pTrc99A cells as described in Step 1 of Experiment 3A and obtain a value of 0.376. Assuming a plasmid copy number of 630 plasmids per cell, and a 100% recovery of plasmid DNA from 3.00 mL of the cells, what is the theoretical yield of plasmid DNA in micrograms (ug)? 2 points (Note: the size of pTrc99A is 4176 base pairs and the average molecular weight of double-stranded DNA is 660 g/mole of base pairs; report your answer with three significant figures)
Please show all work, thank you!
Explanation / Answer
Answer: The theoretical yield of plasmid DNA in micrograms (ug) is 2.600 micrograms.
Calculation:
In general, OD600 value of 1.0 indicates presence of 8.0 x 108 cells per ml.
An OD600 value of 0.376 indicates presence of 8.0 x 108 x 0.376 cells per ml. Which is 3.008 x 108 cells per ml.
From the question 3 ml of cells are used to extract plasmid. Thus total number of cells in 3 ml = 3.008 x 3 x 108 cells.
Which is 9.024 x 108 cells in 3 ml.
Each cell has 630 plasmids.
Assuming 100% recovery, the total number of plasmid molecules extracted from 9.024 x 108 cells (in 3 ml) will be 630 x 9.024 x 108 = 5685.12 x 108 plasmid molecules.
Each plasmid has 4176 base pairs.
Total number of base pairs in 5685.12 x 108 plasmid molecules = 4176 x 5685.12 x 108 base pairs = 23741061.12 x 108 base pairs, which can be written as 2.374 x 107 x 108 = 2.374 x 1015 base pairs.
Molecular weight of dsDNA is 660 g / mole of base pairs. 1 mole is Avagadro number of base pairs, which is 6.022 x 1023 base pairs. 6.022 x 1023 base pairs weigh 660 grams.
If 6.022 x 1023 base pairs is 1 mole, then 2.374 x 1015 base pairs will be (2.374 x 1015)÷ (6.022 x 1023) moles. Which will be 0.394 x 10-8 moles.
If 1 mole of base pairs weigh 660 grams, then 0.394 x 10-8 moles will weigh 0.394 x 10-8 x 660 grams = 260.04 x 10-8 grams
260.04 x 10-8 grams can be written as 2.600 x 102 x 10-8 grams = 2.600 x 10-6 grams.
In other words, 2.600 micrograms.
The theoretical yield of plasmid DNA in micrograms is 2.600 micrograms.
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