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Helicos™ DNA Sample Preparation Manual
Chapter 1
Laboratory Setup
Chapter 2
Sample Preparation
Appendixes
Appendixes
Helicos BioSciences Corporation
One Kendall Square, Building 700
Cambridge, MA 02139 USA
Phone: 877-2-HELICOS
Local: 617-264-1800
Fax: 617-264-1700
D/N LB-010
CONFIDENTIAL
Disclaimer
© Copyright 2009. Helicos BioSciences Corporation. All rights reserved.
No part of this publication may be reproduced, transmitted, transcribed, stored in a retrieval
system, or translated into any language or computer language, in any form, or by any means,
electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written
permission of Helicos BioSciences Corporation. One Kendall Square, Building 700, Cambridge,
MA 02139 USA.
Helicos, HeliScope Single Molecule Sequencer, tSMS, HeliScope Analysis Engine, HeliScope
Sample Loader, Virtual Terminator Nucleotides, and the Helicos logo are trademarks of Helicos
BioSciences Corporation.
BlueJuice and XCell SureLock are trademarks of Invitrogen Corporation.
Covaris is a registered trademark of Covaris, Inc.
NanoDrop is a trademark of Thermo Fisher Scientific Inc.
SYBR is a registered trademark of Molecular Probes, Inc.
MAXYMum is a registered trademark of Axygen Scientific Inc.
Helicos BioSciences Corporation
One Kendall Square, Building 700
Cambridge, MA 02139 USA
Phone: 877-2-HELICOS
Local: 617-264-1800
Fax: 617-264-1700
For Research Use Only. Not for use in diagnostic procedures.
Content subject to change.
CONFIDENTIAL
Revision 2.0
CONFIDENTIAL
Table of Contents
List of Figures
List of Tables
Preface
About this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Chapter 1 Laboratory Setup
DNA Sample Preparation Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimated Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermocycler Programs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Required Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Helicos Provided Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1-2
1-2
1-3
1-3
1-3
Chapter 2 Sample Preparation
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
Step 1: DNA Fragmentation and Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Step 1a: Ultrasonic Shearing of DNA Samples (3 minutes per sample). . . . . . . . . . . . . . . . . . . 2-4
Step 1b: Size Selection using solid phase reversible immobilization (SPRI) (1 hr 30 min) . 2-6
Step 1c: Calculating the Approximate Concentration of 3’ Ends (1 hour 30 minutes). . . . . 2-8
Step 2: PolyA Tailing of the DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10
Step 2a: PolyA Tailing Reaction (30 minutes setup, 1 hour 15 minutes incubation) . . . . . 2-10
Step 2b: Determining the Success of the Tailing Reaction (1 hour 30 minutes) . . . . . . . . . 2-14
Step 2b-1: Short Tail Correction (30 minutes setup, 1 hour 15 minutes incubation) . . . . . 2-17
Step 3: Blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-19
Step 3a: 3’ Blocking Reaction (15 minute setup, 1 hour 15 minutes incubation) . . . . . . . . 2-19
Appendix A Oligonucleotides
Helicos™ PolyA Tailing Control Oligo TR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2
Appendix B Materials Required
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Consumables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B-2
B-3
B-4
B-5
Index
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Rev. 2.0
List of Figures
Figure 2-1: Principles of the Helicos DNA Sample Preparation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2
Figure 2-2: DNA Sample Preparation Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
Figure 2-3: Determine the Average Number of Base Pairs in the Middle of the Sample Smear by Comparing to the Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-9
Figure 2-4: Sample Dilutions Required. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10
Figure 2-5: PolyA Tailed Samples Do Not Migrate Normally in the Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-15
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Rev. 2.0
List of Tables
Table 1-1: Estimated Times Including Preparation and Wait Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
Table 1-2: Thermocycler Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Table 2-1: Required Materials - Step 1a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Table 2-2: Required Materials - Step 1b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
Table 2-3: Required Materials - Step 1c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8
Table 2-4: Required Materials - Step 2a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11
Table 2-5: Sample Tailing Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12
Table 2-6: Volume of Sample DNA to Give 3.0 pmoles of Ends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12
Table 2-7: Control Tailing Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12
Table 2-8: Volume of Sample DNA to Give 0.80 pmoles of Ends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13
Table 2-9: Required Materials - Step 2b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-14
Table 2-10: Decision Tree for Sample Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16
Table 2-11: Required Materials - Step 2b-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-17
Table 2-12: Required Materials - Step 3a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-19
Table B-1: Required Reagents for DNA Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-3
Table B-2: Required Consumables for DNA Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-4
Table B-3: Required Equipment for DNA Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-5
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Rev. 2.0
Preface
About this Manual
This manual provides instructions for setting up and performing the Helicos™ DNA Sample
Preparation. The manual contains two chapters and two appendices.
Chapter 1 - Laboratory Setup - Describes steps required to set up the DNA Sample Preparation.
Chapter 2 - Sample Preparation - Describes the steps required to perform the Helicos™ DNA
Sample Preparation.
Appendix A - Oligonucleotides - Lists oligonucleotides used in the DNA Sample Preparation.
Appendix B - Materials Required - Provides a full listing of all materials required.
For information about how to use the HeliScope™ Single Molecule Sequencer, refer to the
Helicos™ Genetic Analysis System Operator’s Manual (LB-001).
Conventions
This manual uses the following conventions to notify operators of information that requires
special attention. When these symbols appear, observe the appropriate precautions.
Calls attention to a potentially hazardous situation, which, if not
avoided, may result in death or serious injury. It also warns of
chemical hazards.
Calls attention to a potentially hazardous situation, which if not
avoided, may result in minor or moderate injury. This symbol may
also be used to alert against unsafe practices, potential damage to
the instrument, or loss of data.
IMPORTANT
Provides information necessary for proper instrument operation,
sample preparation, and/or sample loading.
NOTE
Provides additional information or tips to aid you in performing
the tasks described in this manual.
Example
Provides an example to help you understand the information
presented in this manual.
Bold type
Bold face is used for emphasis. For example,
Example: Select the Sample button.
Rev. 2.0
CONFIDENTIAL
ix
Related Documentation
Helicos™ Reagents Package Inserts - Provide instructions for handling and storing the Helicos
reagents.
Helicos™ Genetic Analysis System Operator’s Manual (LB-001) - Provides instructions for running
the HeliScope™ Single Molecule Sequencer.
Assumptions
This manual assumes knowledge and experience with basic molecular biology techniques and
standard laboratory safety procedures.
Technical Support
Technical Support for Helicos customers is available via phone and email.
Phone: 1-877-2-HELICOS
support@helicosbio.com
x
CONFIDENTIAL
Rev. 2.0
Chapter 1
Laboratory Setup
Topics described in this chapter include
DNA Sample Preparation Setup
Rev. 2.0
page 1-2
Estimated Times
page 1-2
Thermocycler Programs
page 1-3
Required Materials
page 1-3
Helicos Provided Reagents
page 1-3
CONFIDENTIAL
1-1
DNA Sample Preparation Setup
This section describes DNA Sample Preparation Setup procedures including:
••
••
••
••
Estimated Times
Thermocycler Programs
Required Materials
Helicos Provided Reagents
Estimated Times
Table 1-1: Estimated Times Including Preparation and Wait Times
Assay Step
Minimum Time
Day
Step 1: DNA Fragmentation and Quantitation
Step 1a: Ultrasonic Shearing of DNA Samples
3 minutes per sample
Step 1b: Size Selection using solid phase reversible 1 hour 30 minutes
immobilization (SPRI)
Step 1c: Calculating the Approximate
Concentration of the 3’ Ends
1 hour 30 minutes
Day 1
Step 2: PolyA Tailing of the DNA
Step 2a: PolyA Tailing Reaction
1 hour 45 minutes
Step 2b: Determining the Success of Tailing
Reaction
1 hour 30 minutes
Step 2b-1 (if necessary): Short Tail Correction
1 hour 45 minutes
Step 3: Blocking
Step 3a: 3’ Blocking Reaction
1-2
CONFIDENTIAL
1 hour 30 minutes
Rev. 2.0
Thermocycler Programs
The DNA Sample Preparation uses the programs listed in Table 1-2.
Table 1-2: Thermocycler Programs
Thermocycler Program
Names
Program
Step
hTR_DENAT
1. 95oC 5 minutes
Step 2a: PolyA Tailing Reaction
and
Step 3a: 3’Blocking Reaction
hTR_POLYA
1. 37oC 60 minutes
2. 70oC 10 minutes
Step 2a: PolyA Tailing Reaction
o
3. 4 C forever
hTR_BLOCK
1. 37oC 60 minutes
2. 70oC 10 minutes
Step 3a: 3’Blocking Reaction
3. 4oC forever
Required Materials
The DNA Sample Preparation requires several reagents, general laboratory consumables, and
equipment not supplied by Helicos. Refer to Appendix B, “Materials Required” for a complete
list. We recommend the use of Axygen MAXYMum Recovery tips and tubes or similar for all
storage and sample preparation steps in order to minimize sample loss.
Helicos Provided Reagents
The DNA Sample Preparation also requires the following reagents, which are provided in the
Helicos™ DNA Sample Preparation Reagents Kit:
••
••
••
Helicos™ PolyA Tailing Control Oligo TR
Helicos™ PolyA Tailing dATP
Helicos Control Oligonucleotides
These reagents should be stored at -80oC until ready for use.
Rev. 2.0
CONFIDENTIAL
1-3
1-4
CONFIDENTIAL
Rev. 2.0
Chapter 2
Sample Preparation
Topics described in this chapter include
Introduction
page 2-2
Step 1: DNA Fragmentation and Quantitation
page 2-4
Step 1a: Ultrasonic Shearing of DNA Samples (3 minutes per
sample)
page 2-4
Step 1b: Size Selection using solid phase reversible
immobilization (SPRI) (1 hr 30 min)
page 2-6
Step 1c: Calculating the Approximate Concentration of 3’ Ends (1 page 2-8
hour 30 minutes)
Step 2: PolyA Tailing of the DNA
page 2-10
Step 2a: PolyA Tailing Reaction (30 minutes setup, 1 hour 15
minutes incubation)
page 2-10
Step 2b: Determining the Success of the Tailing Reaction (1 hour
30 minutes)
page 2-14
Step 3: Blocking
page 2-19
Step 3a: 3’ Blocking Reaction (15 minute setup, 1 hour 15 minutes page 2-19
incubation)
Rev. 2.0
CONFIDENTIAL
2-1
Introduction
The Helicos™ DNA Sample Preparation protocol is designed for the amplification-free
modification of DNA samples for sequencing using the Helicos Genetic Analysis System. The
procedure can be used on unamplified DNA purified directly from biological tissue, PCR
amplicons, or DNA from other sources. DNA molecules are sheared by ultrasonication (if necessary), modified with PolyA tails of a controlled length (to allow for
hybridization to capture-primers on the Flow Cell surface), and modified with a dideoxy
nucleotide to prevent extension at the 3' end during the sequencing-by-synthesis reaction (see
Figure 2-1).
Due to the sensitivity of the Helicos Genetic Analysis System, and the universal nature of the
sample preparation protocols, the addition of any type of carrier DNA (e.g. Salmon sperm or Calf
thymus DNA) to the sample at any time during the sample preparation process is strongly
discouraged under most circumstances. Any and all nucleic acid material in the sample will be
modified during the sample preparation process, and could potentially be sequenced by the
HeliScope Sequencer in addition to or instead of the desired sample nucleic acids.
Figure 2-1. Principles of the Helicos DNA Sample Preparation Protocol
2-2
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Rev. 2.0
Figure 2-2 shows an overview of the DNA Sample Preparation. Each step is described in this
chapter.
Sample Preparation
Step 1: DNA Fragmentation and Quantitation
1a: Ultrasonic Shearing of DNA
Samples
1b: Size Selection Using Solid Phase
Reversible Immobilization (SPRI)
1c: Calculating the Approximate
Concentration of 3’ Ends
Step 2: PolyA Tailing of the DNA
2a: PolyA Tailing Reaction
2b: Determining the Success of the
Tailing Reaction
2b-1: Short Tail Correction
Step 3: Blocking
3a: 3’ Blocking Reaction
Figure 2-2. DNA Sample Preparation Overview
Rev. 2.0
CONFIDENTIAL
2-3
Step 1: DNA Fragmentation and Quantitation
Step 1a: Ultrasonic Shearing of DNA Samples (3 minutes per sample)
Ultrasonic shearing of the sample DNA results in its fragmentation, permitting the sequencing
of the entire length of the original sample. This fragmentation procedure has been optimized
for use with amplicons of 300 bp to 10 kb in length, but may be modified for use with shorter
DNA samples.
The protocol makes use of a Covaris S2 instrument equipped with a sample holder for glass
microTubes. Additional information regarding optimization of the shearing conditions to
achieve an average fragment size of 200 bp can be found in the protocol entitled "DNA Shearing
with microTubes (<1.5 kb fragments)" (Part Number 400056, Rev B) on the Covaris website:
http://www.covarisinc.com.
Follow manufacturer's instructions for setting up the S2 instrument as outlined in Operating
conditions in the "DNA Shearing with microTubes (<1.5 kb fragments)" protocol
(http://www.covarisinc.com/pdf/pn_400056.pdf). In brief:
1
2
3
Fill tank with fresh de-ionized water to the proper fill line. The water should cover the
visible part of the glass tube (i.e., to the bottom of the snap-cap).
Degas water for 30 min.
Set the chiller to 4oC. Run the S2 when the temperature software display is between 6o
and 8oC.
Required Materials
Table 2-1: Required Materials - Step 1a
Reagents
Consumables
Equipment
Sample DNA* (1 - 3 µg)
Covaris microTube with AFA
fiber and Snap-Cap
Covaris S2 instrument
Distilled water
200 µL aerosol-free pipette
tips
Covaris Preparation Station
10 X TE, pH 8.0
10 µL aerosol-free pipette tips MicroTube holder (single
sample)
*Starting DNA volume must
not exceed 100 µL volume.
1.5 ml tubes
p-200 pipette
p-10 pipette
Microcentrifuge equipped
with adaptors for 0.2 ml PCR
tubes
Ice bucket
2-4
CONFIDENTIAL
Rev. 2.0
Procedure
(Follow the manufacturer's instructions as outlined in the protocol entitled "DNA shearing with
microTubes" Part Number 400056, Rev B):
1
† Prepare 1 -3 µg of DNA in a final volume of 100 µL of TE.
NOTE: : If the DNA is not in 100 µL of TE, add the appropriate amount of 10 X TE, pH 8.0 and
distilled water to make the overall concentration of TE in the solution 1X.
2
† Place unfilled Covaris microTube into the preparation station holder.
3
† Keeping the cap on the tube, use the p-200 pipette and 200 µL aerosol-free tip to
transfer the 100 µL of DNA sample by inserting the tip through the pre-split septa as
follows:
a
b
With the pipette tip approximately half way down the interior of the tube and
alongside the interior wall, slowly discharge the fluid into the tube.
Be careful not to introduce a bubble into the bottom of the tube. This may happen
if the sample is loaded too quickly.
CAUTION A bubble in the bottom of the tube is in the acoustic field and will deflect
energy, inducing variable results.
CAUTION Do not remove the snap-cap prior to sample processing.
NOTE: The pre-split septa should self-seal after removal of the pipette; be careful not to
pressurize the sample during loading. Rev. 2.0
4
† Keep the microTube in a vertical orientation while transferring it to the S2 microtube
holder. Slide the tube into the holder. Make sure the sample is centered in the holder. Do
not introduce bubbles into the bottom of the tube.
5
† Taking care not to bounce the holder, carefully insert it into the S2 instrument.
6
† Shear the sample for 3 cycles of 60 seconds, 10% duty cycle, 5 Intensity, 200 cycles per
burst.
7
† Remove the tube from the S2 holder and place into the preparation station holder.
Remove the snap cap with the tool supplied with the preparation station. Use the p200
pipette to transfer the sheared DNA to a new, clean 1.5 ml microfuge tube. A brief
centrifugation may be used to collect any DNA remaining in the microTube.
8
† Samples may be stored at -20oC after this step.
CONFIDENTIAL
2-5
Step 1b: Size Selection using solid phase reversible immobilization (SPRI) (1
hr 30 min)
The sample clean-up step uses a modified version of the Agencourt® AMPure® PCR Purification
Protocol to remove small nucleic acids, nucleotides, PCR primers and salts from shearing
reactions.
Required Materials
Table 2-2: Required Materials - Step 1b
Reagents
Consumables
Equipment
AMPure® beads
1000 µL pipette tips
Dynal® Magnet
100% EtOH
200 µL pipette tips
p-1000 pipette
Distilled water
20 µL pipette tips
p-200 pipette
10 µL pipette tips
p-20 pipette
1.5 ml tubes
p-10 pipette
Microcentrifuge
Procedure
1
† Warm the AMPure bead solution to room temperature and vortex thoroughly to resuspend all beads.
IMPORTANT Warm the AMPure beads to room temperature prior to use. This step is
critical!
2
† Prepare 70% EtOH. (7 mL is sufficient for 5 samples)
IMPORTANT Prepare fresh 70% EtOH stock from 100% EtOH stock each time for
optimal results. Do not use a stock 70% EtOH.
3
† Measure the sheared sample volume with a pipette.
4
† Add water to bring each sample to a volume of exactly 100 uL. Vortex the AMPure beads
again and add 300 uL of the AMPure bead slurry to the sample. Example: For a 95 uL
sheared sample volume, add 5 uL of water and 300 uL of AMPure bead slurry.
5
† Incubate the sample slurry for 30 minutes at room temperature.
6
† During this time, shake or flick the tube to mix the solution every 10 minutes.
Alternatively, use a rotisserie to rotate the samples end-to-end during the 30 min
incubation.
7
† After the 30 minute incubation, briefly centrifuge the 1.5 mL tube for 5 seconds in a low
speed microcentrifuge.
8
† Capture the AMPure beads by placing the tube on the Dynal magnet for 5 minutes.
Wash AMPure Beads
9
†
Carefully aspirate the supernatant keeping the tube(s) on the Dynal magnet.
IMPORTANT Do not disturb the beads adhering to the side of the tube. Take care not
to remove any AMPure beads.
10 †
2-6
Add 700 uL of 70% EtOH to each tube on the Dynal magnet.
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Rev. 2.0
11 †
Keeping the tubes on the Dynal magnet, carefully aspirate the supernatant. Repeat
substeps 10-11 one time.
IMPORTANT Do not disturb the beads adhering to the side of the tube. Take care not
to remove any AMPure beads.
12 † Briefly centrifuge the tubes to collect any remaining 70% EtOH at the bottom of the
tube. Place the tubes back on the Dynal magnet and remove the last drop of 70% EtOH
with a 10 uL pipette.
13 † Dry the pellet for 10-15 minutes at room temperature.
NOTE: Remove any visible 70% EtOH droplets from the side of the tubes.
Elution
14 †
Elute the sheared DNA sample from the AMPure beads by adding 20 uL of water to each
tube.
15 †
Pipette the entire volume of the tube up and down 20 times so that the beads are
completely re-suspended to ensure complete elution of the sample from the beads.
16 † Place the tube back on the Dynal magnet with its lid open for 30 minutes.
17 † Collect the 20 uL of supernatant and place it into a new 1.5 mL tube. This supernatant
contains the sheared, size-selected DNA.
18 †
Add another 20 uL of water to the tube.
19 † Repeat the elution by pipetting the entire volume of the tube up and down about 20
times so that the beads are completely re-suspended.
20 † Place the tube with the lid closed back on the Dynal magnet for 5 minutes. Collect the
supernatant and add to the first elute. The final sheared, size-selected DNA volume
should be 40 uL.
Rev. 2.0
CONFIDENTIAL
2-7
Step 1c: Calculating the Approximate Concentration of 3’ Ends (1 hour 30
minutes)
After fragmentation and size selection, the DNA molecules must be modified at their 3' ends
using a tailing procedure, the efficiency of which is determined by the concentration of DNA
ends in the sample. This step (Step 1c) allows the proper estimation of the concentration of 3'
ends, by taking into account both the average length of the DNA fragments, assessed by gel
electrophoresis, and the total DNA concentration in the sample.
Required Materials
Table 2-3: Required Materials - Step 1c
Reagents
Consumables
Equipment
SYBR® Gold Nucleic Acid Gel
Stain
10 µL pipette tips
Photodocumentation System
compatible with a SYBR® Gold
photographic filter
Distilled water
SYBR® Gold photographic
filter
10X BlueJuice™ gel loading
buffer
NanoDrop™ 1000 or 8000
spectrophotometer
Ultrapure 10X TBE buffer
XCell SureLock™ Mini-Cell or
equivalent electrophoresis
apparatus compatible with 10 X 10 cm gel cassettes
25 bp DNA ladder
p-10 pipette
1 kb DNA ladder
p-2 pipette
4-20% TBE gel 1.0 mm, 12-well
or similar
Procedure
To calculate the approximate concentration of 3’ ends:
1
† Determine the average size of the sheared DNA by running an aliquot on a 4-20% TBE
gel 1.0 mm, 12-well or similar, as follows:
a
b
c
d
e
f
2-8
Load 2 µL aliquots of all samples in 1X BlueJuice™ DNA loading buffer in a total
volume of 10 µL.
Load 1 µL of a 1:10 dilution of a 1 kb and a 25 bp ladder in 1X BlueJuice™ buffer in a
total volume of 10 µL.
Run gel in 1X TBE buffer (180 volts for 45 minutes).
Stain for 10 minutes in SYBR® Gold Nucleic Acid Gel Stain diluted 1:10,000 in water.
Destain in water for 10 - 15 minutes, changing the water every 2 minutes.
Image with a photodocumentation system compatible with a SYBR® Gold
photographic filter under nonsaturating conditions.
CONFIDENTIAL
Rev. 2.0
g
Determine the average size of your sample (in base pairs) by comparing the size of
the middle of the sample smear to the size standards (see Figure 2-3).
1: 1 kb DNA ladder
1
2
3
2: Sheared sample
3: 25 bp DNA ladder
(~200 bp)
NOTE: Bands of unsheared DNA may be visible in the gel.
The middle of the smear of sheared DNA should be used to
determine the average MW.
Figure 2-3. Determine the Average Number of Base Pairs in the Middle of the Sample Smear by Comparing to the Size Standards
2
† Determine the double-stranded DNA concentration at this step using a NanoDrop™
1000 or 8000 spectrophotometer.
3
† Calculate the pmoles of the ends in the sample using the following formula:
pmol ends/µL = XX ng DNA/µL X (103 pg/ng) X (pmole/660 pg) X (1/average # bp as determined from gel photo) X 2 ends/molecule
Sample Calculation:
10 ng DNA/µL X (103 pg/ng) X(pmole/660 pg) X (1/200) X 2 ends/molecule = 0.15 pmol ends/µL
Rev. 2.0
CONFIDENTIAL
2-9
Step 2: PolyA Tailing of the DNA
Step 2a: PolyA Tailing Reaction (30 minutes setup, 1 hour 15 minutes
incubation)
The fragments must be modified at their 3'ends with a PolyA tail to allow for efficient
hybridization onto the oligonucleotide-coated Helicos™ Flow Cell. The length of the PolyA tail
is crucial for proper hybridization and subsequent sequencing. As such, the efficiency of the
tailing reaction is monitored by running a parallel tailing control reaction for every sample. The
latter consumes a small amount of the original sample and includes a precise amount of the
Helicos™ PolyA Tailing Control Oligo TR, which will be analyzed by gel electrophoresis and
assessed for tailing efficiency in Step 2b of the protocol.
IMPORTANT Two Master Mixes are required; one for the Sample tailing reaction (see Table 2-5), and one for the Control reaction (see Table 2-7).
IMPORTANT Two Sample dilutions are required; one (containing 3.0 pmole of sample) for the
Sample tailing reaction and one (containing 0.8 pmole of sample) for the Control reaction (see
Figure 2-4).
Figure 2-4. Sample Dilutions Required
For each DNA sample, you will be preparing a Sample reaction that will be sequenced. In
addition, you will be preparing a Control reaction to determine the success of your trailing
reaction.
2-10
CONFIDENTIAL
Rev. 2.0
Required Materials
Table 2-4: Required Materials - Step 2a
Reagents
Consumables
Equipment
Terminal Transferase Kit
Ice water
Prechilled Aluminum Block
milled for 0.2 mL tubes
10 µL pipette tips
p-10 pipette
20 µL pipette tips
p-20 pipette
200 µL pipette tips
p-200 pipette
0.2 mL PCR tubes
Thermocycler
1.5 ml tubes
Ice Bucket
••
••
••
Terminal Transferase
Enzyme (20 U/µL)
CoCl2 (2.5 mM)
Terminal Transferase 10X
Buffer
Helicos™ DNA Sample
Preparation Reagents Kit
••
••
Helicos ™ PolyA Tailing
Control Oligonucleotide
TR
Helicos™ PolyA Tailing
dATP
Distilled water
Microcentrifuge with adaptors
for 0.2 ml PCR tubes (or strip
tube rotor)
Procedure
To perform PolyA tailing reactions:
NOTE: Prechill the Aluminum Block to 0oC in an ice and water slurry. This will prevent the reannealing of the denatured, single-stranded DNA products.
1
† Prepare the master mix for the 3.0 pmole Sample reactions as shown in Table 2-5.
IMPORTANT Do not substitute generic dATP for the Helicos PolyA Tailing dATP, keep
this and all other reagents on ice during preparation of the master mixes. Keep master
mixes on ice. Do not re-use the Helicos PolyA Tailing dATP for subsequent reactions.
NOTE: Master mix volumes include a 10% scale-up
Rev. 2.0
CONFIDENTIAL
2-11
Table 2-5: Sample Tailing Master Mix
Reagent
1X
Terminal Transferase 10X Buffer
4.4 µL
22.0 µL
CoCl2 (2.5 mM)
4.4 µL
22.0 µL
Terminal Transferase Enzyme (20 U/µL)
2.2 µL
11.0 µL
Helicos™ PolyA Tailing dATP
4.2 µL
21.0 µL
Distilled water
1.3 µL
6.5 µL
16.5 µL
82.5 µL
Total
2
5X
† Based on the calculations in Step 1c, determine the volume of sample DNA that would
give 3.0 pmoles of ends and dilute as shown in Table 2-6.
Table 2-6: Volume of Sample DNA to Give 3.0 pmoles of Ends
3
DNA Volume for 3.0 pmoles
x µL
Distilled water
(25 - x) µL
† Prepare the master mix for the 1pmole Control reactions as shown in Table 2-7.Master
mix volumes include a 10% scale-up.
Table 2-7: Control Tailing Master Mix
Reagent
1X
Terminal Transferase 10X Buffer
4.4 µL
22.0 µL
CoCl2 (2.5 mM)
4.4 µL
22.0 µL
Terminal Transferase Enzyme (20 U/µL)
2.2 µL
11.0 µL
Helicos™ PolyA Tailing dATP
1.4 µL
7.0 µL
Distilled water
4.1 µL
20.5 µL
16.5 µL
82.5 µL
Total
4
2-12
†
5X
Based on the calculations in Step 1c, determine the volume of sample DNA that would
give 0.80 pmoles of ends and dilute as shown in Table 2-8
CONFIDENTIAL
Rev. 2.0
Table 2-8: Volume of Sample DNA to Give 0.80 pmoles of Ends
DNA Volume for 0.80 pmoles
x µL
Helicos™ PolyA Tailing Control
Oligo TR
1 µL
Distilled water
25 - (x+1) µL
5 †Prepare a separate Oligo TR control reaction (without sample DNA), by putting 4 μL of
Helicos PolyA Tailing Control Oligo TR and 21 μL of distilled water into a 0.2 ml PCR tube
6 †Before adding the master mixes, place the 3.0 and 0.80 pmole tubes (prepared in Steps
2 and 4 above) in the thermocycler and run the hTR_DENAT program. After 5 minutes,
immediately remove the DNA tubes from the thermocycler and snap cool for a minimum of
2 minutes by placing in an Aluminum Block (milled for 0.2 mL tubes) that has been prechilled
in ice water.
hTR_DENAT program (95oC for 5 min)
IMPORTANT It is essential to chill the block to 0oC in an ice and water slurry, and cool
the sample as quickly as possible to 0oC to prevent the re-annealing of the denatured,
single-stranded DNA products. 7
† Add 15 µL of the Sample Tailing Master Mix to each Sample DNA tube.
IMPORTANT Mix by pipetting up and down thoroughly 10 times. The mixing step is
crucial to the success of the tailing reaction.
8
† Add 15 µL of the Control Tailing Master Mix to each Control tube.
IMPORTANT Mix by pipetting up and down thoroughly 10 times. The mixing step is
crucial to the success of the tailing reaction.
9
† Collect the contents of the tubes into the bottom of the tubes by briefly centrifuging.
10 † Place the tubes in the thermocycler and run the hTR_POLYA program.
hTR_POLYA program (37oC for 60 min, 70oC for 10 min, 4oC forever)
NOTE: This is an optional stopping point in the protocol. Samples should be stored at -20oC.
Rev. 2.0
CONFIDENTIAL
2-13
Step 2b: Determining the Success of the Tailing Reaction (1 hour 30
minutes)
To ensure efficient hybridization to the Helicos Flow Cell, the PolyA tail lengths at the ends of
the DNA fragments must fall within an optimal size range. The Control reactions, which were
processed in the previous step (Step 2a), serve as proxies for the success of the tailing reaction.
In this procedure, the Control reactions are analyzed by gel electrophoresis, staining and
visualization, which allows the detection of the Helicos™ PolyA Tailing Control Oligonucleotide
TR. The length of this tailed control oligonucleotide, as assessed by gel electrophoresis,
provides an accurate measurement of PolyA tail length.
Required Materials
Table 2-9: Required Materials - Step 2b
Reagents
Consumables
Equipment
Distilled water
10 µL pipette tips
Photodocumentation System
compatible with a SYBR® Gold
photographic filter
4-20% TBE gel 1.0 mm, 12-well
or similar
SYBR® Gold photographic
filter
10X BlueJuice™ gel loading
buffer
XCell SureLock™ Mini-Cell or
equivalent electrophoresis
apparatus compatible with 10
cm X 10 cm gel cassettes
100 bp DNA ladder
p-10 pipette
SYBR® Gold Nucleic Acid Gel
Stain
p-2 pipette
Ultrapure 10X TBE Buffer
2-14
CONFIDENTIAL
Rev. 2.0
Procedure
To determine the success of the tailing reactions:
1
† Run 20 µL of the Control reactions on a 4-20% TBE gel 1.0 mm, 12-well or similar, as
follows. Retain the remaining 20 µL to use in Step 2b-1(if needed).
a
b
c
d
e
f
Load 20 µL aliquots of the Control reaction in 1X BlueJuice™ DNA loading buffer
(18 µL of sample and 2 µL of 10X BlueJuice™).
Load 1 µL of a 1:10 dilution of a 100 bp ladder in 1X BlueJuice™ buffer in a total
volume of 20 µL.
Run gel in 1X TBE buffer (180 volts for 45 minutes).
Stain for 10 minutes in SYBR® Gold Nucleic Acid Gel Stain diluted 1:10,000 in water.
Destain in water 10 - 15 minutes, changing water every 2 minutes.
Image with a photodocumentation system compatible with a SYBR® Gold
Photographic Filter under nonsaturating conditions (see Figure 2-5).
1: 100 bp DNA Ladder
2: 200 dA Control
3: 90 dA Control
4: 90 dA Tailing Control Oligo TR
5: 25 bp DNA Ladder
NOTE: Tailed oligos with 90 to 200 dA are expected to migrate below
the 600 bp band to midway between the 200 and 300 bp bands on the
100 bp ladder. Lane 2 contains a Control Reaction with 200 dA. Lane 3
contains a Control Reaction band with 90 dA. Lane 4 contains the tailed
Helicos PolyA Tailing Control Oligo TR.
Figure 2-5. PolyA Tailed Samples Do Not Migrate Normally in the Gel
Rev. 2.0
CONFIDENTIAL
2-15
Table 2-10: Decision Tree for Sample Migration
Sample Migration*
PolyA Tail Length*
Next Step
Between 250 and 600 bp
~90 to 200 nt
Proceed to Step 3a: 3’ Blocking
Reaction (15 minute setup, 1
hour 15 minutes
incubation) on page 2-19.
Below 250 bp
< 90 nt
Proceed to Step 2b-1: Short
Tail Correction (30 minutes
setup, 1 hour 15 minutes
incubation) on page 2-17.
Above 600 bp size marker.
Sample material not limited.
> 200 nt
**Repeat Step 2: PolyA Tailing
of the DNA on page 2-10 using
twice the amount of sheared
DNA.**
Above 600 bp size marker.
Limited sample material.
> 200 nt
Proceed to Step 3a: 3’ Blocking
Reaction (15 minute setup, 1
hour 15 minutes
incubation) on page 2-19.
NOTE: May result in an
excessive number of PolyA
reads during sequencing.
*PolyA tailed samples do not migrate normally on the gel.
** All Control samples should migrate at the size of the Oligo TR Control sample. A longer PolyA
Tail may be indicative of a sample with a reduced number of DNA strands ending in a 3’ OH. Only
strands ending in a 3’ OH are able to be tailed.
2
† If the band in the Control reaction lane migrates anywhere between 250 bp and 600 bp,
the sample is properly PolyA tailed (i.e., the tail length is >90 and <200 dA in length).
Proceed to Step 3a: 3’Blocking Reaction.
NOTE: PolyA tailed samples do not migrate normally in the gel (see Figure 2-5). 3
† If the band in the Control reaction lane migrates under 250 bp, the sample has a PolyA
tail shorter than 90 nt. In this case, proceed to Step 2b-1: Short Tail Correction below.
IMPORTANT If proceeding to Step 2b-1, use a new aliquot of Helicos™ PolyA Tailing
dATP for the Sample and Control reactions. 4
2-16
† If the band in the Control reaction lane migrates above 600 bp, the PolyA tail contains
more than 200 dA. In this case, the sample may be run on the Helicos™ Genetic Analysis
System; however, if sample is not limiting, we recommend repeating the PolyA tailing
(Step 2a, substeps 1-5) on another sheared DNA aliquot using twice the amount of input
DNA to generate a sample with the optimal length PolyA tail.
CONFIDENTIAL
Rev. 2.0
Step 2b-1: Short Tail Correction (30 minutes setup, 1 hour 15 minutes
incubation)
Samples that exhibit short tails during Step 2b can be corrected by re-extending their tails.
IMPORTANT This step is not required unless your samples exhibit short tails in Step 2b.
Required Materials
Table 2-11: Required Materials - Step 2b-1
Reagents
Consumables
Equipment
Terminal Transferase Enzyme
(20U/µL)
Ice Water
Thermocycler
Helicos™ PolyA Tailing dATP
10 µL pipette tips
Ice Bucket
Prechilled Aluminum Block
milled for 0.2 mL tubes
p-10 pipette
p-2 pipette
Microcentrifuge with adaptors
for 0.2 ml PCR tubes (or strip
tube rotor)
Procedure
To perform Short Tail Correction:
1
† Place both the Sample reactions and the corresponding Control reactions in the
thermocycler and run the hTR_DENAT program.
hTR_DENAT program (95oC for 5 min)
2
† After 5 minutes, immediately remove tubes from the thermocycler and snap cool for a
minimum of 2 minutes by placing in an Aluminum Block milled for 0.2 mL tubes that has
been prechilled in ice water.
IMPORTANT It is essential to chill the block to 0oC in an ice and water slurry and cool
the sample as quickly as possible to 0oC to ensure complete denaturation.
3
† For Sample reactions.
••
Add 3.9 µL of dATP and 2 µL of terminal transferase.
IMPORTANT Mix by pipetting up and down thoroughly 10 times.
4
† For Control reactions:
••
Prepare a 1:2 dilution of the dATP stock.
(For example 5 µL 50 µM dATP + 5 µL distilled water.)
••
Add 1.3 µL of diluted dATP stock and 1 µL of terminal transferase.
IMPORTANT Mix by pipetting up and down thoroughly 10 times.
5
Rev. 2.0
† Collect the contents of the tubes into the bottom of the tubes by centrifuging briefly.
CONFIDENTIAL
2-17
2-18
6
† Place the DNA tubes in the thermocycler and run the hTR_POLYA program.
hTR_POLYA program (37oC for 60 min, 70oC for 10 min, 4oC forever)
7
† Repeat Step 2b, substeps 1-4 on the Control reactions to determine if the PolyA tail
length is now greater than 90 dA.
CONFIDENTIAL
Rev. 2.0
Step 3: Blocking
Step 3a: 3’ Blocking Reaction (15 minute setup, 1 hour 15 minutes
incubation)
During hybridization, the PolyA tails on the modified templates may align imperfectly to the
oligonucleotides on the Helicos Flow Cell surface. This may result in the generation of a
recessed 3' end that can serve as a substrate for the sequencing-by-synthesis reaction. To
prevent the incorporation of fluorescent Virtual Terminator™ nucleotides at that end of the
duplexes, the 3' ends of the template molecules must be modified with a dideoxy terminator.
Only the Sample reactions undergo the blocking step.
NOTE: Blocking results in a one base pair shift. Such a shift would not be detectable on a gel run on
the Control reactions prepared above. For this reason, the Sample reactions are the only reactions that
are blocked.
Required Materials
Table 2-12: Required Materials - Step 3a
Reagents
Consumables
Equipment
Distilled Water
Ice water
Prechilled Aluminum Block
milled for 0.2 mL tubes
Terminal Transferase Enzyme 10 µL pipette tips
(20U/µL)
10 µL pipette
500 µM Biotin ddATP diluted 20 µL pipette tips
1:1 from PerkinElmer NEL-548
(1mM)
20 µL pipette
200 µL pipette tips
200 µL pipette
Thermocycler
Microcentrifuge with adaptors
for 0.2 ml PCR tubes (or strip
tube rotor)
Rev. 2.0
CONFIDENTIAL
2-19
Procedure
To perform the 3’ Blocking Reaction:
1
† Following Step 2a, the PolyA Tailing Reaction, place the tubes in the thermocycler and
run the hTR_DENAT program. After 5 minutes of incubation at 95oC, immediately
remove the DNA tubes from the thermocycler and snap cool for a minimum of 2
minutes by placing in an Aluminium Block milled for 0.2 ml tubes that has been
prechilled in ice water.
hTR_DENAT program (95oC for 5 min)
2
† Add 0.3 µL of 500 µM Biotin ddATP to each tube.
3
† Add 2 µL of the Terminal Transferase enzyme to each tube.
IMPORTANT Mix thoroughly by pipetting up and down 10 times. The mixing step is
critical to the success of the 3' Blocking Reaction step.
4
† Collect the contents of the tubes into the bottom of the tubes by briefly centrifuging.
5
† Place the tubes back into the thermocycler and run the hTR_BLOCK program.
hTR_BLOCK program (37oC for 60 min, 70oC for 10 min, 4oC forever
NOTE: Store samples at -20oC until ready for downstream processing.
2-20
CONFIDENTIAL
Rev. 2.0
Appendix A
Oligonucleotides
Topics described in this chapter include
Helicos™ PolyA Tailing Control Oligo TR
Rev. 2.0
CONFIDENTIAL
page A-2
A-1
Helicos™ PolyA Tailing Control Oligo TR
5'-TCCACTTATCCTTGCATCCATCCTCTGCCCTG-3'
A-2
CONFIDENTIAL
Rev. 2.0
Appendix B
Materials Required
Topics described in this chapter include
Rev. 2.0
Introduction
page B-2
Required Reagents
page B-3
Consumables
page B-4
Equipment
page B-5
CONFIDENTIAL
B-1
Introduction
This appendix lists all materials (required reagents, consumables, and equipment) that are required for the DNA Sample Preparation. This information is also provided in each section of the
document where the procedures are described.
B-2
CONFIDENTIAL
Rev. 2.0
Required Reagents
Table B-1: Required Reagents for DNA Sample Preparation
Reagents
Vendor
Catalog Number
10 X TE, pH 8.0
Invitrogen
15581-044
4-20% TBE gel 1.0 mm, 12-well or similar
Invitrogen
EC62252BOX
AMPure® beads
Agencourt
A29152
10X BlueJuice™ gel loading buffer
Invitrogen
10816-015
25 bp DNA ladder
Invitrogen
10597-011
1 kb DNA ladder
Invitrogen
15615-016
SYBR® Gold Nucleic Acid Gel Stain
Invitrogen
S11494
Ultrapure 10X TBE Buffer
Invitrogen
15581-044
100 bp DNA ladder
Invitrogen
15628-019
Terminal Transferase Kit
NEB
M0315L
PerkinElmer
NEL-548001
Distilled Water
••
••
••
Terminal Transferase Enzyme (20U/µL)
CoCl2
Terminal Transferase 10x
1 mM Biotin-11- ddATP
Rev. 2.0
CONFIDENTIAL
B-3
Consumables
Table B-2: Required Consumables for DNA Sample Preparation
Consumables
Vendor
Catalog Number
Covaris microtube with AFA fiber and Snap-Cap
Covaris
Part #520045
0.2ml MAXYMum Recovery Thin Wall PCR
Tubes
Axygen
PCR-02-L-C
1000 µL MAXYMum Recovery Filter Tips
Axygen
TF-1000-L-R-S
200 µL MAXYMum Recovery Filter Tips
Axygen
TF-200-L-R-S
20 µL MAXYMum Recovery Filter Tips
Axygen
TF-20-L-R-S
0.1-10 µLMAXYMum Recovery Micro Tips
Axygen
TXLF-10-L-R-S
1.5 ml tubes MAXYMum Recovery
Axygen
MCT-150-LC
Ice Water
B-4
CONFIDENTIAL
Rev. 2.0
Equipment
Table B-3: Required Equipment for DNA Sample Preparation
Equipment
Vendor
Catalog Number
Corvaris S2 Instrument
Covaris
Part # 500003
Preparation Station
Covaris
Part # 500142
microTube holder (single sample)
Covaris
Part # 500111
Aluminum Block milled for 0.2 ml tubes
VWR
13259-260
XCell Surelock Mini-Cell Electrophoresis
Apparatus or equivalent
Invitrogen
E1001
SYBR Gold Photographic Filter
Invitrogen
S7569
NanoDrop Spectrophometer
ThermoScientific
NanoDrop 1000
or 8000
p1000 pipette
p200 pipette
p10 pipette
p2 pipette
Thermocycler
Microcentrifuge
Ice Bucket
Photodocumentation system compatible
with a SYBR Gold Photographic filter
Dynal® Magnet
Rev. 2.0
CONFIDENTIAL
B-5
B-6
CONFIDENTIAL
Rev. 2.0
Index
Numerics
3’ blocking reaction, 2-19
A
Assumptions, x
B
Blocking, 2-19
Blocking reaction
3’, 2-19
C
Calculating concentration of 3’ ends, 2-8
Concentration of 3’ ends
calculating, 2-8
Consumables, B-4
Contacting
technical support, x
Conventions
text, ix
Correction
short tail, 2-17
D
Determining success of tailing reaction, 2-14
DNA
fragmentation, 2-4
PolyA tailing of, 2-10
quantitation, 2-4
DNA Sample Preparation Setup, 1-2
DNA samples
ultrasonic shearing, 2-4
Documentation
related, x
P
PolyA tailing of DNA, 2-10
PolyA tailing reaction, 2-10
Preparation time
estimated, 1-2
Primers, A-1
Principle of operation
TR, 2-2
Programs
thermocycler, 1-3
R
Reactions
PolyA tailing, 2-10
Reagents
Helicos provided, 1-3
required, B-3
Related documentation, x
Required
reagents, B-3
Required materials, 1-2, 1-3
S
Sample preparation, 2-2
3’ blocking reaction, 2-19
blocking, 2-19
calculating concentration of 3’ ends, 2-8
determining success of tailing reaction, 2-14
DNA fragmentation and quantitation, 2-4
PolyA tailing of DNA, 2-10
PolyA tailing reaction, 2-10
short tail correction, 2-17
size selection, 2-6
ultrasonic shearing of DNA samples, 2-4
Short tail correction, 2-17
Size selection, 2-6
Support
contacting, x
T
Helicos provided reagents, 1-3
Tailing reaction
determining success, 2-14
Technical support
contacting, x
Text conventions, ix
Thermocycler programs, 1-3
Time, TR preparation, 1-2
O
U
E
Equipment, B-5
Estimated preparation times, 1-2
H
Overview, 2-3
Rev. 2.0
Ultrasonic shearing of DNA samples, 2-4
CONFIDENTIAL
CONFIDENTIAL
Rev. 2.0