240
- 75 μL of freshly prepared 70% ethanol should be added (com-
bine 230 μL of ethanol with 100 μL of nuclease-free water per
sample) and the tube should be moved from side to side in the
magnet to wash the beads, then the supernatant is discarded
without disturbing the pellet. - The steps should be repeated for a second wash.
- Ensure that all ethanol droplets are removed from the wells.
Keeping the plate in the magnet, air-dry the beads at room
temperature for 5 min. Do not overdry (see Note 3). - The constructed libraries can then be stored at − 20 °C for
future template preparation. - Proceed to library quantification.
- We used Experion 1K DNA kit (Fig. 2 ). It can also be quanti-
fied by qPCR Qubit™ 2.0 or 3.0 Fluorometer, or with the
Agilent™ 2100 Bioanalyzer™ instrument. - Eleven samples can be analyzed on the chip at a time. Samples
should be grouped into pools according to previous barcoding
and according to experimental design. - For each pool, all samples should be diluted to the same con-
centration (10 ng/μL) and then equal volumes should be
added to a single tube and calculations should be done accord-
ing to the manual. - Based on the calculated library concentration, determine the
dilution that results in a concentration of ~100 pM.
3.1.2 Library
Quantification
Fig. 2 Automated electrophoresis system with electrophoresis machine (E), prim-
ing station (P), and vortex station (V). It helps in quantification of prepared library.
It is an integrated system, which incorporates electrophoresis, staining, destain-
ing, band detection, and imaging into a single stepSuja Pillai et al.