User guide

74
Fragment Library Preparation Using the AB Library Builder
™
System: 5500 Series SOLiD
™
Systems User Guide
AppendixE Checklist and workflow tracking form
Workflow checklists: prepare a standard or express fragment library with the AB Library Builder
™
System Kit
E
Equipment Reagents Preparation steps
Set up the AB Library
Builder™ System
 Protocol Card
 Cartridge Rack
 Tip and Tube Rack
 AB Library Builder
™
System
 Library Builder
™
Fragment
Core Kit for 5500 SOLiD™
 AMPure
™
XP Reagent Kit
 Library Builder
™
Fragment
Core Kit for 5500 SOLiD™
 5500 SOLiD
™
Fragment
Library Standard Adaptors or
5500 SOLiD
™
Fragment
Library Barcode Adaptors
 Thaw cartridges
completely.
 Add Agencourt
AMPure
®
XP Reagent to
cartridges.
 Add library adaptors to
Ligation Buffer Tubes.
 Load all tubes, tips, and
cartridges into the AB
Library Builder
™
Device.
Nick-
translate,
then
amplify the
library
 Thermal cycler
 PCR strip tubes
 AB Library Builder
™
Amplification Reagents
 Thaw Library PCR
Primers 1 and 2 on ice.
 Thaw Platinum
™
PCR
Amplification Mix on
ice.
Quantitate
the library
 Real-time PCR
system
 SOLiD
™
Library TaqMan
®
Quantitation Kit
—
(Optional)
Pool
equimolar
libraries of
similar size
— — —
(Optional)
Gel-purify
the libraries
 iBase
™
System
 E-gel
®
Safe
Imager™ instrument
 E-Gel
™
2% SizeSelect
™
gel
 50-bp DNA Ladder
 Nuclease-free Water
 Thaw 50 bp DNA
Ladder on ice.
(Optional) P
Pool
remaining
libraries to be
combined into
a single
emulsion
—
— —
Shear the DNA
 Covaris
®
S220
System
 Covaris microTube
adaptor
 Covaris microTube
loading station
 Covaris
®
microTube
 1× Low TE Buffer
 Shear Buffer
 Ethylene glycol
 Degas the water in the
Covaris
®
S220 System
30 minutes prior to use.
 Supplement the
circulated water chiller
with 20% ethylene
glycol.
 Thaw Shear Buffer at
room temperature.