Cost-efficient Yeast genome Flongle library
Yutaro Hori
Abstract
A cost-efficient protocol for constructing a Flongle library.
Steps
Assemble the following components to end repair and add A to the genomic sample:
| A | B |
|---|---|
| Ultra II buffer | 1.75 |
| End repair buffer | 1.75 |
| Ultra II enzyme | 1 |
| End repair enzyme | 1 |
| DNA | x (500 ng) |
| MQ | 44.5 - x |
Incubate at 20°C for 0h 5m 0s, then 65°C for 0h 5m 0s.
Add 50µL of Ampure beads (or equivalent beads) and mix well.
Wash with 75 % EtOH twice and elute with 11µL of 10millimolar (mM) Tris-HCl (pH 8.0).
Keep the tube on the magnetic rack and do not disturb the beads while washing with 75% EtOH.
Use 1µL of the solution to check the concentration with Qubit. The concentration needs to be at least 30ng/uL.
Assemble the following components for ligation:
| A | B |
|---|---|
| DNA | 10 uL |
| TLB | 4 uL |
| Quick ligase | 1.5 uL |
| AMX | 0.8 uL |
Incubate at Room temperature for 0h 30m 0s.
Add 2.3µL of 5Molarity (M) NaCl and incubate at Room temperature for 0h 30m 0s and then cfg at max speed for 0h 20m 0s.
Remove sup and add PEG wash buffer, cfg at max speed for 0h 1m 0s .
Remove sup and add 11µL of 10mM Tris-HCl (pH 8.0) and incubate at 4°C for.
Check the concentration with Qubit.
Assemble the following priming solution:
| A | B |
|---|---|
| FLB | 2.5 uL |
| FB | 97.5 uL |
and the library mix solution:
| A | B |
|---|---|
| DNA | x uL (100 ng) |
| SB II | 10 uL |
| LB II | 4 uL |
| MQ | 6 - x uL |
. Load them and start sequencing.
