Quality Check for the extrcated DNA, quantifying NGS libraries.

Author:

Vinay K L

Copyright:

None

Date:

06/10/2022

Purpose

To quantify the concentration of extracted DNA and NGS libraries.

Two methods of quantification

Nanodrop

We do not have this in our lab, but if required, use the ones in Ram’s lab (Nanophoto meter) or the one in Ashwani Sharma’s lab at Chemistry department.

Steps

  1. Wash the NanoDrop pedestal with distilled water and use only KimWipes to clean the lower pedestal.

  2. Set the blank using 2ul of the solution DNA is suspended in. This is usually buffer AE (Kit method of extraction) or TE (PCI method of extraction).

  3. Make sure to wipe both the pedestals vigorously to clean.

  4. Add 2ul of your sample to the lower pedestal and then lower the upper arm.

  5. After ~15 seconds, readings will be displayed, make sure to note down concentration, A260/280, A230/260 ratio.

  6. Clean both the pedestals with water and wipe it clean with KimWipes.

Qubit

Qubit is the preferred method of DNA quantification due to its sensitivity.

  1. Take out the working solution from fridge and let it sit at room temperature for ~15 minutes.

  2. Turn on the Qubit machine and select DNA -> 1X HS High sensitivity option.

  3. Check the last date of calibration. If the last calibration is older than a week, make sure to calibre the machine with Standards.

  4. To calibre, add 190ul of working solution and 10ul of Standard ‘#1’ and Standard ‘#2’ and vortex for 15 seconds. Incubate the tubes at room temperature for about 2 minutes.

  5. Once calibrated, add 198ul of working solution to each Qubit tubes per sample and 2ul of sample and vortex. Incubate for 2 minutes.

  6. Note down the concentration readings and discard the tubes.