User guide

14
Fragment Library Preparation Using the AB Library Builder
™
System: 5500 Series SOLiD
™
Systems User Guide
Chapter2 Prepare to Build the Library
Quantitate the DNA
Quantitate the DNA
For accuracy, determine sample DNA concentration using a double-stranded DNA-
specific fluorescence assay. Use the HS Assay Kit to measure dsDNA concentrations
from 10 pg/µL to 100 ng/µL. For samples outside this range, use the dsDNA BR for
higher concentrations of DNA or PicoGreen
®
dsDNA Assay Kit for lower
concentrations:
•Invitrogen Qubit
™
dsDNA HS Assay Kit (Invitrogen Part no. Q32851 or Q32854)
or
•Invitrogen Qubit
™
dsDNA BR Assay Kit (Invitrogen Part no. Q32850 or Q32853).
or
• Invitrogen Quant-iT
™
PicoGreen
®
dsDNA Assay Kit (Invitrogen Part no. P7589)
Shear the DNA
This step involves fragmenting the input DNA into small fragments with a mean
fragment size of 165 bp by using the Covaris
®
System. The conditions have been tested
for shearing 10 ng–5 µg DNA in a total volume of 120 µL. For certain DNA samples,
optimizing the shearing protocol may be necessary.
Shear the DNA
You can shear the DNA with two supported shearing systems:
•The Covaris
®
S220 System (see “Shear the DNA with the Covaris
®
S220 System”).
or
•The Covaris
®
S2 System (see “Shear the DNA with the Covaris
®
S2 System” on
page 15.
Shear the DNA with the Covaris
®
S220 System
IMPORTANT! Ensure that the bath temperature during shearing is between
5–10°C. Higher shearing temperatures can be harmful to DNA.
1. For each library dilute the components below in a 1.5-mL LoBind Tube. Shear
Buffer reduces DNA damage from fragmentation.
2. Prepare the Covaris
®
S220 Tank:
a. Ensure that the water in the Covaris
®
S220 tank is filled with fresh deionized
water to fill-line level 12 on the graduated fill-line label.
The water should cover the visible glass part of the tube.
Component Amount
DNA 10 ng–5 µg
1✕ Low TE Buffer Variable µL
Shear Buffer 1.2 µL
Total 120 µL