Question

1. A student uses the same gel extraction and quantification protocols used in Lab 5, with the following exceptions:

   1. Only half of the 50µL pMD2 plasmid digestion reaction was loaded on the gel.

   2. The student did a 1:25 dilution of the extracted backbone during the quantification.

The student measures the same absorbance (0.015). Using the protocol form the lab manual, what was the concentration (in ng/µL) of the plasmid stock concentration before performing the digestion reaction? (2pts)

Gel extraction Materials PE buffer QG buffer Isopropanol • Spin columns • Scalpel Methods • Pipet solutions in the middle of the membrane of the spin Note: All centrifugation steps are carried out at 17.900 g column, not on the side/wall of (13,000 rpm) at room temperature. the spin column Don't 1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel into a clean 2ml tube. . Forget to tare the scale with an A maximum of 400mg of gel is acceptable. empty 1.5mL tube before 2. Weigh the gel slice in a microcentrifuge tube. Add Add weighing your gel slice 3 volumes of Buffer QG to 1 volume of gel • Touch the membrane of the spin (100mg is approximately 100ml). column with your pipet tip • Pipet-mix when inside the spin 3. Incubate at 50°C for 10 min or until the gel slice 50°C for 10 min or until the del slice column as the membrane is very has completely dissolved. To help dissolve gel, fragile mix by vortexing the tube every 2-3 min during the incubation. 4. After the gel slice has dissolved completely, check that the color of the mixture is yellow.

Answer 1

Result of DNA quantification: Initial plasmid volume is 3 uL and mixed in PCR grade water 297 uL (1:100; 1 part DNA and 99 parts PCR grade water), so final dilution factor would be 100 in the DNA sample

So, dilution factor (DF) is =100

Place all values in formula

OD A260 is 0.015

Dilution factor is 0.015

"Molar attenuation coefficient" is 50

DNA concentration =0.015x100x50

DNA concentration = 75 ng/uL

For Digestion

Fifty microliter pMD2 plasmid used for digestion and out of which half of volume was used for gel loading that is 25 uL,

DNA samples were further diluted and in this case DF was 25, coefficient is 50 and absorbance is 0.015

So, DNA concentration would be

=0.015x25x50= 18.75 ng/uL

So DNA concentration in the extracted backbone is 18.75ng/uL

So the concentration of the extracted DNA was 75 ng/uL and this was the stock concentration before digestion.