User guide
32
Fragment Library Preparation Using the AB Library Builder
™
System: 5500 Series SOLiD
™
Systems User Guide
Chapter4 Nick-Translate the Library with Optional Amplification
(Optional) Nick-translate and amplify the library
6. Run the PCR for each 125-µL aliquot:
Purify the nick-
translated,
amplified 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 amplified 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.
Stage Step Temp Time
Holding Nick translation 72°C 20 min
Holding Denature 95°C 5 min
Cycling Denature 95°C 15 sec
Anneal 62°C 15 sec
Extend 70°C 1min
Holding Extend 70°C 5 min
Holding — 4°C ∞
Component Volume
Nuclease-Free Water 600 µL × N
Ethanol, Absolute 1400 µL × N
Total 2000 µL × N
Component Volume
Nick-translated and amplified library 500 µL
Agencourt AMPure
®
XP Reagent 750 µL
†
† Equal to 1.5 volumes of sample reaction.