User guide

33
Fragment Library Preparation Using the AB Library Builder
™
System: 5500 Series SOLiD
™
Systems User Guide
Chapter4 Nick-Translate the Library with Optional Amplification
Quantitate the DNA
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.
e. Transfer the supernatant containing the amplified library to a new 1.5-mL
LoBind Tube.
Quantitate the DNA
Measure the DNA concentration by using:
• 2 µL of sample with the Qubit
™
dsDNA HS Assay Kit (Invitrogen Part
no. Q32851) and the Qubit
®
2.0 Fluorometer (Invitrogen Part no. Q32866). Use the
Qubit
™
dsDNA HS Assay Kit to measure dsDNA concentrations from 10 pg/µL
to 100 ng/µL. For samples outside this range, use the Qubit
™
dsDNA BR Assay
Kit for higher concentrations of DNA or the Invitrogen Quant-iT
™
PicoGreen
®
dsDNA Assay Kit for lower concentrations
or
• 2 µL of sample in the NanoDrop
®
ND-1000 Spectrophotometer (see “Quantitate
the DNA with the NanoDrop
®
ND-1000 Spectrophotometer” on page 55)
or
• 1 µL of sample in the Agilent Technologies 2100 Bioanalyzer
™
. If you used the
bioanalyzer, see “Check the size distribution of the library” on page 34.
and/or
• The appropriate volume in qPCR [refer to the Applied Biosystems SOLiD
™
Library
TaqMan
®
Quantitation Kit protocol (Invitrogen Part no. A12120)]
STOPPING POINT Store the DNA in Elution Buffer (E1) at 4°C for short-term storage or at
–20°C for long-term storage. Proceed directly to emulsion PCR [refer to the SOLiD
™
EZ Bead
™
Emulsifier Getting Started Guide (Part no. 4441486)] or “Check the size
distribution of the library” on page 34.