User guide

30
Fragment Library Preparation Using the AB Library Builder
™
System: 5500 Series SOLiD
™
Systems User Guide
Chapter4 Nick-Translate the Library with Optional Amplification
Nick-translate the library
Purify the nick-
translated library
1. Resuspend the Agencourt AMPure
®
XP Reagent and allow the mixture to come
to room temperature (~30 minutes).
2. Prepare 70% ethanol for N number of libraries:
3. For every nick-translated library, label a new 1.5-ml LoBind Tube.
4. Combine each set of the identical 4 PCR reactions (125 µL) to the appropriately
labeled 1.5-mL LoBind Tube. The total combined volume of the amplified library
is 500 µL.
5. Bind the DNA to the resuspended, ambient Agencourt AMPure
®
XP Reagent:
a. For each library, prepare the bead suspension:
b. Vortex the beads for 10 seconds, then pulse-spin.
c. Incubate the mixture at room temperature (20–25°C) for 5 minutes.
d. Place each tube in a DynaMag
™
-2 Magnetic Rack for at least 3 minutes until
the solution is clear of brown tint when viewed at an angle; then, remove and
discard the supernatant.
6. Wash the DNA 2 times. For each wash:
a. Without removing the tube from the magnet, add 750 µL of freshly prepared
70% ethanol and incubate for 30 seconds. Do not disturb the pellet.
b. Aspirate and discard ethanol.
7. Remove the tube from the DynaMag
™
-2 Magnetic Rack, pulse-spin the tube,
return the tube to the magnetic rack; then remove and discard the supernatant
with a 20-µL pipettor.
8. Open each tube, then dry the beads at room temperature (20–25°C) for
≤ 5minutes.
9. Elute the DNA:
a. Remove each tube from the DynaMag
™
-2 Magnetic Rack, then add 50–
100 µL Low TE Buffer directly to the pellet to disperse the beads.
b. Vortex the beads for 10 seconds, then pulse-spin.
c. Incubate the beads for 2 minutes at room temperature.
d. Place the tube in the DynaMag
™
-2 Magnetic Rack for at least 1 minute until
the solution clears.
Component Volume
Nuclease-Free Water 600 µL × N
Ethanol, Absolute 1400 µL × N
Total 2000 µL × N
Component Volume
Nick-translated library 500 µL
Agencourt AMPure
®
XP Reagent 750 µL
†
† Equal to 1.5 volumes of sample reaction.