Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Users Guide
WARNING
For continued protection against fire hazard, replace fuses with those of the same
type and rating.
AVERTISSEMENT
Remplacez les fusibles par ceux de mme type et puissance pour viter les risques
dincendie.
WARNING
Most of the reagents and solvents used in PNA and nucleic acid synthesis are
hazardous. Wear a lab coat, gloves and eye protection when handling reagents.
Adequate ventilation is essential and working under a fume hood is recommended.
Consult Appendix A, Reagent Safety, for reagent safety considerations.
AVERTISSEMENT
La plupart des ractifs et solvants employs en synthse PNA en d'acides
nucliques sont dangereux. Portez une blouse, des gants et des lunettes de
protection lorsque vous manipulez des ractifs. Une ventilation adquate est
ncessaire et il est recommand de travailler sous une hotte. Consultez la partie
Appendix A, Reagent Safety, de ce manuel.
EMC
This device complies with Part 15 of the FCC Rules. Operation is subject to the following two
conditions: (1) This device may not cause harmful interference, and (2) this device must accept
any interference received, including interference that may cause undesired operation.
Warning: Changes or modifications to this unit not expressly approved by the party responsible
for compliance could void the users authority to operate the equipment.
Note: This equipment has been tested and found to comply with the limits for a Class A digital
device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable
protection against harmful interference when the equipment is operated in a commercial
environment. This equipment generates, uses, and can radiate radio frequency energy and, if not
installed and used in accordance with the instruction manual, may cause harmful interference to
radio communications. Operation of this equipment in a residential area is likely to cause harmful
interference in which case the user will be required to correct the interference at his own expense.
Note: Shielded cables must be used with this unit to ensure compliance with the Class A FCC
limits.
Scurit
Cet instrument a t vrifi avec la norme C22.2 No. 151, Spcifications de scurit du matriel
lectrique utilis pour les mesures, les contrles et dans les laboratoires ; Partie 1 : Spcifications
gnrales, et il est conforme cette norme. Cest un produit homologu par les ETL Testing
Laboratories.
EMC
This Class A digital apparatus meets all requirements of the Canadian Interference-Causing
Equipment Regulations.
Cet appareil numrique de la classe A respecte toutes les exigences du Rglement sur le
materiel brouilleur du Canada.
Manufacturers Name:
Applied Biosystems
Manufacturers Address:
Type of Equipment:
Laboratory Instrumentation
Part Number:
GEN600041 or GEN600042
Serial Number:
FX???8200?
Question marks represent date and manufacturing codes that are part of the serial number.
Year of Manufacture:
Table of Contents
Table of Contents
How to Use This Guide ............................................................................ ix
Chapter 1 Introduction to PNA Synthesis
1.1
1.2
1.3
1.4
......................................... 1-2
PNA Synthesis on the Expedite 8900 System ................................ 1-5
Characteristics of PNA Oligomers ............................................... 1-7
Guidelines for Sequence Design of PNA Oligomers ........................ 1-10
1.4.1
1.4.2
1.4.3
2.1
Overview
2.2
Overview
vii
Table of Contents
4.1
4.2
4.3
4.4
4-2
4-2
4-3
4-6
4.4.1
4.4.2
4.4.3
4.4.4
4.5
4.5.1
4.5.2
5.2
viii
Applied Biosystems
Chapter 1,
Introduction to PNA
Synthesis
Chapter 2,
Installing the PNA
Software
Chapter 3,
Performing a
Synthesis
Chapter 4,
Post-Synthesis
Procedures
Chapter 5,
Troubleshooting and
Maintenance
Appendix A,
Reagent Safety
Appendix B,
Ordering Information
ix
Appendix C,
PNA Reference
Information
Appendix D,
Technical Support
and Training
Related
documents
Conventions
General
conventions
Notes, Cautions,
and Warnings
Applied Biosystems
CAUTION
Changing reagent bottles during a synthesis is not
recommended. Check the reagent resources prior to
initiating a synthesis to make sure that there is a sufficient
supply.
A warning calling out information essential to the safety of the
operator appears as:
WARNING
The Expedite Cabinet weighs 102 pounds (46 kg). Two
people are required to safely lift the instrument cabinet.
Send us your
comments
xi
1Introduction to PNA
Synthesis
1-1
Chapter 1
N-terminal
HO
H3N
O
P
N
H
-O
N
O
O
O
N
H
O
P
n
-O
N
O
NH2
HO
C-terminal
3'-end
PNA
PB100697
DNA
PB100698
1-2
Applied Biosystems
Expedite PNA
monomers
N
H
OH
PB100692
Monomer
O
O
HN
N
HN
N
PB100694
PB100693
N
C monomer
A monomer
MW=701.73
MW=724.75
O
O HN
O N N
H
HN
O
PB100695
N
PB100696
G monomer
T monomer
MW=741.76
MW=506.51
1-3
Chapter 1
1
O
O
N
H
O-spacer monomer
MW=384.41
OH
O
O
PB100691
Activation and
coupling
1-4
Applied Biosystems
Protocol
1-5
Chapter 1
Overview of PNA
synthesis
Wash
DMF
Cleave and
deprotect
Cap
acetic anhydride
lutidine in DMF
3 minutes
Fmoc deblock
20% piperidine
in DMF
2.5 minutes
Wash
DMF
Wash
DMF
Activate and Couple
5 eq DIPEA 7.5 eq lutidine
5 eq monomer 4.5 eq HATU
in NMP/DMF (1/2)
8.25 minutes
1-6
Applied Biosystems
Thermal stability
Thermal stability
Stronger binding independent of salt concentration
Greater sequence specificity
Strand invasion
Resistance to nucleases and proteases
1-7
Chapter 1
Tm
15-mer PNA/DNA
69C
15-mer DNA/DNA
54C
15-mer PNA/RNA
72C
15-mer DNA/RNA
50C
Stronger binding
independent of
salt concentration
1-8
Applied Biosystems
PNA/DNA
Tm
DNA/DNA Tm
0 mM
72C
38C
140 mM
69C
56C
1000 mM
65C
65C
Reprinted with permission of Nature (1993, 365, 565567). PNA and DNA
15-mers were hybridized to a complementary DNA 15-mer in
10mM phosphate buffer, pH7, with varying salt concentrations.
Greater
specificity of
interaction
Strand invasion
Resistance to
nucleases and
proteases
1-9
Chapter 1
General guidelines
Triplex formation
Strand invasion
DNA 5'
PNA
3'
H2NOC
NH2
C-terminal
N-terminal
1-10
Applied Biosystems
Length of PNA
oligomer
Purine content
*Notice that the complement, the probe for the other strand
includes a good design sequence:
GAA CCT GCT ATC TAG
1-11
Chapter 1
Labels
Label
Example
Labeled at N-Terminal
Fluorescein
Flu-O-O-PNA
Biotin
Bio-O-O-PNA
Label at C-Terminal
Fluorescein
Acetyl-PNA-O-Lys(Flu)
1-12
Applied Biosystems
Bio-O-O-PNA-O-Lys(Flu)
Sequences to
avoid
Homo thymine
PNA oligomers
Stability
NH2
CONH2
3'
5'
CONH2
NH2
1-13
Chapter 1
Cytosine content
Unanticipated
triplex formation
Ionic strength
1-14
Applied Biosystems
PNA clamps
PNA-clamp
dsDNA
PB100656
1. Egholm, M. et al. Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Research 23, 217222 (1995).
2. Griffith, M.C. et al. Single and Bis Peptide Nucleic Acids as triplexing agents: Binding and
stoichiometry. Journal of the American Chemical Society 117, 831832 (1995).
3. Veselkov, A.G., Demidov, V.V., Nielsen, P.E. & Frank-Kamenetskii, M.D. A new class or
genome rare cutters. Nucleic Acids Research 24, 24832487 (1996).
1-15
2-1
Chapter 2
2.1 Overview
The PNA instrument boot disk includes:
PNA
DNA
RNA
Thio
User
CAUTION
Follow the proper flushing procedures when switching
between PNA reagents and standard nucleic acid reagents.
Switching reagents without proper flushing can damage the
instrument. See Section 3.7, Loading PNA Reagents, for
more information.
2-2
Applied Biosystems
2.
Remove the old instrument disk from the drive (the disk
drive is located at the upper left rear of the instrument).
3.
4.
5.
6.
2-3
Chapter 2
Expedite
workstation
software
2-4
Applied Biosystems
3Performing a Synthesis
3-1
Chapter 3
Performing a Synthesis
3.1 Overview
Key steps in a
synthesis
2.
3.
4.
5.
6.
7.
8.
9.
10.
3-2
Applied Biosystems
2.
3.
4.
3-3
Chapter 3
Performing a Synthesis
6.
DNA
RNA
THIOATE
USER
PNA
7.
8.
9.
3-4
Applied Biosystems
10.
11.
Direction of
synthesis
3-5
Chapter 3
Performing a Synthesis
Reagent kit
Wash A (DMF)
Wash B (DMF)
Deblocking solution
Capping solution
Base solution
3-6
Applied Biosystems
Preparing
activator
Preparing
monomers
1.
2.
Preparing amino
acids
1.
Preparing
Fmoc-AEEA-OH
spacer
Columns
3.
1.
2.
3-7
Chapter 3
Performing a Synthesis
Flushing before
switching to PNA
reagents
3-8
Applied Biosystems
2.
3.
4.
5.
6.
Remove all the bottles, replace with clean dry bottles and
press Ok. At the end of the dryout routine, the instrument
beeps ten times to indicate the shutdown procedure is
finished.
7.
8.
9.
10.
Flushing before
switching to
standard nucleic
acid reagents
2.
3.
4.
5.
6.
Remove all the bottles, replace with clean dry bottles and
press Ok. At the completion of the dryout routine, the
instrument beeps ten times to indicate it is done with the
shutdown procedure.
3-9
Chapter 3
Performing a Synthesis
7.
8.
9.
10.
11.
3-10
Applied Biosystems
Turning off
blanket gas
Bottle locations
2.
Top
rack
A
6
G
7
WshB
1
T
8
Cap
2
9
WshA
WA7*
Dblk
3
Bottom
rack
AuxC
DB8*
AuxB
4
AuxA
5
AuxA
6
External caddy
*Labels on reagent lines
3-11
Chapter 3
Performing a Synthesis
3-12
Applied Biosystems
Reagent
Bottom row
1
Wash solvent B
Capping solution
Deblocking solution
46
Top row
A, C, G, T
Monomers
Fmoc-AEEA-OH (O-spacer)
67
Base solution
Activator solution
External reagents
2 L bottle
(rear)
1 L bottle
(front)
2 L bottle
(not shown)
Waste container
3-13
Chapter 3
Performing a Synthesis
Resetting bottle
volumes
After loading reagents, use the Bottle Change Tool to reset the
reagent bottle volume:
1.
2.
3.
3-14
Applied Biosystems
Priming
3.8 Priming
Prime the fluidics system after:
Loading reagents
The instrument has been idle for more than 48 hours
Command
Reagent
Pulses*/Volume
Prime
Individual
Selected
Reagent
20 pulses (320 L)
Prime All
Monomers
10 pulses (160 L)
Activator, Caps
and Auxiliary
Internal Wash
30 pulses (480 L)
Wash A and
Deblock
Prime
Reagents
3-15
Chapter 3
Performing a Synthesis
Command
Prime
Monomers
Reagent
Monomers
Pulses*/Volume
10 pulses from each
monomer bottle
followed by 20 pulses of
Activator, 30 pulses of
Internal Wash and a gas
flush.
Priming after
loading reagents
1.
CAUTION
Do not install the column before you prime the reagent
passages. The priming procedure pumps all the
reagents through the column to waste, and would
damage the column.
Union
3-16
Applied Biosystems
Priming
2.
3-17
Chapter 3
Performing a Synthesis
Priming after
48-hour idle
period
1.
CAUTION
Do not install the column before you prime the reagent
passages. The priming procedure pumps all the
reagents through the column to waste, and would
damage the column.
2.
Select Column 1.
A message is displayed instructing you to replace the
column with a union.
5.
3-18
Applied Biosystems
6.
7.
8.
3
Stop
Instrument Stop Button
Stop button
3-19
Chapter 3
Performing a Synthesis
Monitoring the
synthesis
3
Figure 3-9 Synthesis Status Screen
Interrupting the
synthesis
3-20
Applied Biosystems
4Post-Synthesis
Procedures
Removing the
Final Fmoc Protecting Group ................... 4-2
4.2
4.3
4.4
Analysis, Purification,
Handling, and Quantitation....................... 4-6
4.5
4-1
Chapter 4
Post-Synthesis Procedures
2.
3.
2.
3.
4-2
Applied Biosystems
WARNING
TFA is a highly corrosive acid. TFA may be fatal if
swallowed.
AVERTISSEMENT
Lacide trifluoractique (TFA) est un acide hautement
corrosif. Le TFA peut tre mortel sil est aval.
Preparing
cleavage mixture
Scale
Volume of cleavage
mixture required for
each cleavage
2 mole
400 L
25 mole
2.5 mL
4-3
Chapter 4
Post-Synthesis Procedures
Cleaving and
deprotecting
2.
3.
4.
5.
6.
7.
8.
9.
4-4
Applied Biosystems
4-5
Chapter 4
Post-Synthesis Procedures
PNA analysis
Reversed-phase purification of PNA oligomers
PNA handling
PNA quantitation
1.
2.
3.
Determining the
crude yield
2.
3.
4.
AU260 x 500
4-6
Applied Biosystems
Condition
Setting
Column
Eluent A
Eluent B
Gradient
Flow
1.2 mL/min
Temperature
Detector
260 nm
Injection
volume
25 L
Reequilibration
between
runs
5 minutes in 95% A, 5% B
HPLC analysis
2 mL/min
The retention time for the test 10-mer sequence AGT CCA
TTG C is 4 to 6 minutes.
4-7
Chapter 4
Post-Synthesis Procedures
Setting
Column
Eluent A
Eluent B
Gradient
0 to 5% B over 3 minutes
5 to 20% B over 15 minutes
20 to 95% B over 3 minutes
Flow
7.5 mL/min
Temperature
4-8
Detector
Injection
volume
Sample loop
500 L or larger
Applied Biosystems
Purification
1.
2.
3.
NOTE: Do not use the same speed vac for PNA and
DNA or RNA. The small amount of TFA in the PNA
solutions can destroy the DNA or RNA.
4.
Determining
amount of PNA in
fractions
2.
3.
4.
AU260 x 500
4-9
Chapter 4
Post-Synthesis Procedures
Used directly
Divided into aliquots and used or dried on a speed vac
Using purified
PNA
4-10
Applied Biosystems
C=6.6
T=8.6
A=13.7 mLmol-1cm-1
Melting
experiments
4-11
Chapter 4
Post-Synthesis Procedures
Preparing the
reporter group
solution
1.
4-12
Applied Biosystems
Amount
DMF
DIEA
7.7 mg
300 L
6.6 L
10.7 mg
300 L
11 L
Amount
DMF
DIEA
12.0 mg
300 L
11 L
succinimidyl ester
Molecular Wt.
Residue
Mass
N-Hydroxysuccinimido
Biotin
341.40
227.31
5 & 6 Carboxyfluorescein,
succinimidyl ester
473.39
359.3
5 & 6 Tetramethyl
Rhodamine,
527.53
414.46
succinimidyl ester
2.
3.
4.
4-13
Chapter 4
Post-Synthesis Procedures
1.
2.
3.
4.
Fluorescein or rhodamine-labeled
oligomers60 minutes
5.
6.
Cleavage of the
labeled PNA
oligomer from the
support
4-14
Applied Biosystems
At the N-terminal
Via a lysine incorporated into the sequence
4-15
Chapter 4
Post-Synthesis Procedures
NOTE: If you have removed the final Fmoc group and have
not labeled it with another label, labeling will occur at this
position as well as at the lysine side-chain. Avoid this by
capping the N-terminal amino group.
It is important to note that labels at the N-terminal require two
spacer molecules to separate the label from the PNA
oligomer. When a label is to be introduced via a lysine, the
lysine side-chain itself acts as one spacer. The spacer
molecule(s) allows for more efficient attachment of the label,
resulting in higher yields.
Solution-phase
labeling of PNA
oligomers
4-16
Applied Biosystems
2.
3.
4.
5.
Thiol-specific
labels
4-17
5Troubleshooting and
Maintenance
5.2
5-1
Chapter 5
5.1 Troubleshooting
Table 5-1 PNA Troubleshooting
Symptom
PNA does not
precipitate
Mass spectrometry
analysis data shows
the expected MW
+222.2 Da, and HPLC
shows a late eluting
peak
5-2
Applied Biosystems
Possible Cause
Corrective Action
Reagent empty
Insufficient gas
pressure
Blockage in a line
Fmoc protecting
group not removed
Sequence
aggregates
Sequence is
insoluble
Fmoc protecting
group not removed
Troubleshooting
Possible Cause
Corrective Action
Fmoc protecting
group not removed
5
Expedite 8900 PNA Chemistry Users Guide
5-3
Chapter 5
5.2 Maintenance
Proper attention to maintenance will result in increased
instrument life and performance. Regular preventive
maintenance will help the Expedite Nucleic Acid System
perform to specifications.
Daily
Weekly
Monthly
As needed
General
guidelines
5
5-4
Applied Biosystems
AReagent Safety
WARNING
Most reagents and solvents used in PNA synthesis are
hazardous. Wear a lab coat, gloves, and eye protection
when handling reagents. Adequate ventilation is
essential and working under a fume hood is
recommended. Flammable reagents should be kept in
an appropriate flameproof cabinet.
AVERTISSEMENT
La plupart des ractifs et solvants employs en
synthse PNA sont dangereux. Portez une blouse, des
gants et des lunettes de protection lorsque vous
manipulez des ractifs. Une ventilation adquate est
ncessaire et il est recommand de travailler sous une
hotte.
Waste disposal
A-1
Appendix A
Reagent Safety
Acetic Anhydride
A-2
Applied Biosystems
Activator (ACT)
WARNING
Activator may cause allergic skin reaction. Depending on
the intensity of exposure, effects may vary from mild to
severe irritation. Avoid all contact. Use only in a fume hood.
Wear gloves when handling and wash thoroughly after
handling.
AVERTISSEMENT
Lactivateur peut entraner des ractions allergiques sur la
peau. Lirritation peut tre soit lgre, soit svre, selon
lintensit de lexposition. viter tout contact. Utilisez
seulement avec une hotte. Portez des gants lors de la
manipulation et lavez-vous entirement aprs.
A white powder. Should be considered hazardous as the
toxicological properties are unknown. Do not breathe the
powder.
Base solution
(Base)
Capping solution
(CAP)
A-3
Appendix A
Reagent Safety
Cleavage and
deprotection
solution (AP)
Deblock Solution
(Dblk)
Dichloromethane
(DCM) (Methylene
Chloride)
A-4
Applied Biosystems
N,N-diisopropylethylamine (DIEA)
A-5
Appendix A
Reagent Safety
Dimethylformamide (DMF)
2,6-Lutidine
A-6
Applied Biosystems
1-Methyl-2pyrrolidinone
(NMP)
A-7
Appendix A
Reagent Safety
Spill or Leak Procedures: Evacuate the area. Wear selfcontained breathing apparatus and full protective clothing.
Take up with sand or vermiculite and place in a closed
container.
A
Monomer Diluent
Monomers
(A,C,G,T)
Piperidine
A-8
Applied Biosystems
Trifluoroacetic
Acid (TFA)
A-9
Appendix A
Reagent Safety
A
Wash Solutions
(WshA, WshB)
A-10
Applied Biosystems
BOrdering Information
Quantity
Part Number
1 kit
GEN063018
0.508 g
GEN063014
0.493 g
GEN063015
0.519 g
GEN063016
0.355 g
GEN063017
15 mL
GEN063104
0.932 g
GEN063080
15 mL
GEN063100
200 mL
GEN063101
Reagents
B-1
Appendix B
Ordering Information
Quantity
Part Number
200 mL
GEN063102
1L
GEN910003
200 mL
GEN063103
3 pkg
(4/pkg)
GEN063053
Columns
Column, 2 mol Fmoc-XAL PEG-PS
Quantity
Part Number
0.193 g
GEN063032
100 mL
GEN910004
Dichloromethane (DCM)
1 liter
GEN902017
4/pkg
GEN063051
1/pkg
GEN063054
Quantity
Part Number
Filters, endline
set of 19
GEN210168
1 kit
GEN210165
1 kit
GEN210164
1 kit
GEN210167
B-2
Applied Biosystems
Recommended
vendors for
additional
materials
Documentation
m-CresolAldrich C8572-7
GEN601318
GEN601308
B-3
CPNA Reference
Information
C.2
C.3
C.4
C-1
Chapter C
C
Example
2.
3.
Sequence:
(N-C) Bio-O-O-AGT-CCA-TTG-C
Average Masses:
MW3220.21 Da
(M+H)+3221.21 Da
C-2
Applied Biosystems
Volume of crude:
0.20 mL
OD:
100
Composition
MW
275.2720
251.2468
291.2586
266.2586
Total Bases
1.
10
O-spacer
145.16
Amino Acids
varies
Labels
varies
2.
Number of
Residues
3.
C-3
Chapter C
Column
Gradient
Flow
(ml/min)
Reequilibration
Time (min)
1.2
1.0
15
Column
Gradient
ReFlow
equilibration
(ml/min)
Time (min)
7.5
1.5
10
C-4
Applied Biosystems
MALDI-TOF Methods
Preparing
standard and
sample
1.
2.
3.
Description
M+1
Product peak
M+23
Sodium adduct
M+63
Copper adduct
M+222.2
Fmoc-On
M-251
C-deletion
M-266
T-deletion
M-275
A-deletion
M-291
G-deletion
C-5
Chapter C
Description
M+43
Capped sequence
M+16
Oxidation of biotin
C-6
Applied Biosystems
Entry Error
+9
A entered instead of T
-9
T entered instead of A
+15
T entered instead of C
-15
C entered instead of T
+16
G entered instead of A
-16
A entered instead of G
+24
A entered instead of C
-24
C entered instead of A
+25
G entered instead of T
-25
T entered instead of G
+40
G entered instead of C
-40
C entered instead of G
MALDI-TOF Methods
Mass (Da)
A residue
275.27
C residue
266.25
G residue
291.27
T residue
251.24
O Spacer residue
145.16
16
Fmoc
223
Acetyl
43
C-7
Chapter C
N,N-Dimethylformamide
(DMF)
92.6
N-Methylpyrolidinone (NMP)
1.6
Piperidine
4.9
N,N-Diisopropylethylamine
(DIEA)
0.04
2,6-Lutidine
0.25
Acetic Anhydride
0.2
HATU
0.2
0.2
C-8
% Composition
Applied Biosystems
D.2
D.3
D-1
Appendix D
By E-mail
Product/Product Area
E-Mail Address
galab@appliedbiosystems.com
pcrlab@appliedbiosystems.com
Biochromatography
PerSeptive DNA, PNA and Peptide Synthesis
systems
FMAT 8100 HTS System
CytoFluor 4000 Fluorescence Plate Reader
Mariner Mass Spectrometers
Voyager Mass Spectrometers
Mass Genotyping Solution 1 (MGS1) System
tsupport@appliedbiosystems.com
LC/MS
(Applied Biosystems/MDS Sciex)
support@sciex.com
Chemiluminescence (Tropix)
tropix@appliedbiosystems.com
D-2
Applied Biosystems
By telephone or
fax (North
America)
Product/Product Area
Telephone
Fax
1.800.831.6844, then
press 8a
1.650.638.5981
DNA Synthesis
1.800.831.6844, press 2,
then press 1a
1.650.638.5981
1.800.831.6844, press 2,
then press 2a
1.650.638.5981
1.800.831.6844, press 2,
then press 3a
1.650.638.5981
1.800.831.6844, press 2,
then press 4a
1.650.638.5981
1.800.831.6844, press 2,
then press 6a
1.650.638.5981
Peptide Synthesis
(433 and 43 x Systems)
1.800.831.6844, press 3,
then press 1a
1.650.638.5981
Protein Sequencing
(Procise Protein Sequencing
Systems)
1.800.831.6844, press 3,
then press 2a
1.650.638.5981
D-3
Appendix D
Product/Product Area
PCR and Sequence Detection
Telephone
1.800.762.4001, then
press:
Fax
1.240.453.4613
1 for PCRa
2 for TaqMan applications
and Sequence Detection
Systems including ABI
Prism 7700, 7900, and
5700a
6 for the 6700 Automated
Sample Prep Systema
or
1.800.831.6844, then
press 5a
Voyager MALDI-TOF
Biospectrometry Workstations
1.800.899.5858, press 1,
then press 3b
1.508.383.7855
Biochromatography
(BioCAD, SPRINT, VISION, and
INTEGRAL Workstations and
POROS Perfusion Chromatography
Products)
1.800.899.5858, press 1,
then press 4b
1.508.383.7855
1.800.899.5858, press 1,
then press 5b
1.508.383.7855
1.800.899.5858, press 1,
then press 5b
1.508.383.7855
D-4
Applied Biosystems
Product/Product Area
Telephone
Fax
1.800.899.5858, press 1,
then press 5b
1.508.383.7855
1.800.899.5858, press 1,
then press 6b
1.508.383.7855
Chemiluminescence (Tropix)
1.800.542.2369
(U.S. only),
or 1.781.271.0045c
1.781.275.8581
LC/MS
(Applied Biosystems/MDS Sciex)
1.800.952.4716
1.508.383.7899
By telephone or
fax (outside North
America)
Region
Telephone
Fax
27 11 478 0411
27 11 478 0349
33 1 69 59 85 00
27 11 478 0349
27 11 478 0411
D-5
Appendix D
Region
Middle Eastern Countries and North
Africa (Monza, Italia)
Telephone
Fax
61 3 9730 8600
61 3 9730 8799
China (Beijing)
86 10 64106608 or
86 800 8100497
86 10 64106617
Hong Kong
91 11 653 3743/3744
91 11 653 3138
Korea (Seoul)
82 2 5936470/6471
82 2 5936472
60 3 79588268
603 79549043
Singapore
65 896 2168
65 896 2147
Thailand (Bangkok)
66 2 719 6405
Austria (Wien)
43 (0)1 867 35 75 00
43 (0)1 867 35 75 11
Belgium
Europe
420 2 35364314
Denmark (Naerum)
45 45 58 60 00
45 45 58 60 01
Finland (Espoo)
France (Paris)
33 (0)1 69 59 85 85
33 (0)1 69 59 85 00
Germany (Weiterstadt)
Hungary (Budapest)
Italy (Milano)
39 (0)39 83891
Norway (Oslo)
47 23 12 06 05
47 23 12 05 75
D-6
Applied Biosystems
Region
Telephone
Fax
48 22 866 4020
Portugal (Lisboa)
351.(0)22.605.33.14
351.(0)22.605.33.15
Russia (Moskva)
385 1 34 91 927/838
385 1 34 91 840
34.(0)91.806.1210
34.(0)91.806.12.06
Sweden (Stockholm)
Switzerland (Rotkreuz)
31 (0)180 392400
31 (0)180 392409 or
31 (0)180 392499
44 (0)1925 825650
44 (0)1925 282502
44 (0)1925 282481
44 (0)1925 282509
81 3 5566 6230
81 3 5566 6507
52 55 35 3610
52 55 66 2308
Brazil
55 11 5070 9694/95
Argentina
55 11 5070 9694/95
Chile
55 11 5070 9694/95
Uruguay
55 11 5070 9694/95
Japan
Japan (Hacchobori, ChuoKu, Tokyo)
Latin America
D-7
Appendix D
Through the
Technical Support
web site
1.
2.
3.
4.
D-8
Applied Biosystems
Ordering
documents by
telephone
2.
Obtaining
documents
through the
web site
2.
3.
D-9
Appendix D
4.
Right-click
, then select Save Target As to
download a copy of the PDF file.
Select the Fax check box, then click Deliver
Selected Documents Now to have the document
faxed to you.
Select the Email check box, then click Deliver
Selected Documents Now to have the document
(PDF format) e-mailed to you.
D-10
Applied Biosystems
D-11
Index
A
Acetic anhydride, safety
precautions A-2
Activation and coupling, Fmoc 1-4
Activator (ACT), safety precautions A-3
Activator, preparing 3-7
Aggregation, cause of 1-11, 1-13
Amino acid, preparing 3-7
Analysis of PNA 4-6
Applied Biosystems
world wide web address xi
C
Capping solution (CAP), safety
precautions A-3
Certificates of analysis
obtaining D-9
Chemicals, ordering B-1
Chemistry
available in version 2.2 2-2
default, changing 3-4
E
E-mail
contacting technical support D-2
E-mail address, Technical
Publications xi
Extinction coefficients, PNA 4-11
Index-1
I
N
D
E
X
I
N
D
E
X
F
Field Service in North America,
contacting D-3
Fluorescein
and purine content 1-12
labeling PNA with 4-15
removing from oligomer 3-6
Flushing requirements 3-8
Fmoc
definition 1-3
removing 4-2
Fmoc-AEEA-OH spacer
function 3-6
number required for labeling 1-12
preparing 3-7
H
Handling PNA 4-10
HATU, safety precautions A-3
Hazard information, reagents A-1
Holding a synthesis 3-20
Homo thymine oligomers 1-13
How to Use This Guide ix
HPLC methods C-4
I
Installing software 2-2
Instrument stop key 3-19
Interrupting a synthesis 3-20
Ionic strength 1-14
L
Labeling
guidelines 1-12
number of Fmoc-AEEA-OH spacer
Index-2
Applied Biosystems
required 1-12
PNA in solution 4-15
sequences, and purine
content 1-12
support-bound PNA 4-12
Length, PNA oligomer 1-11
Loading reagents 3-11
Lutidine, safety precautions A-6
M
Maintenance 5-4
MALDI-TOF methods C-5
Melting experiments 4-11
Molecular weights of monomers 1-3,
1-4
Monomers
diluent, safety precautions A-8
Expedite PNA 1-3
extinction coefficients 4-11
molecular weights 1-3, 1-4
ordering B-1
preparing 3-7
safety precautions A-8
MSDSs, obtaining D-9
N
N,N- Diisopropylethylamine, safety
precautions A-5
NMP, safety precautions A-7
O
Oligomer, PNA, see PNA
Optical density, conversion to
concentration or weight C-2
Ordering information B-1
Orientation 1-10
O-spacer, see Fmoc-AEEA-OH spacer
P
Part numbers B-1
Piperidine, safety precautions A-8
PNA
analysis 4-6
characteristics of 1-7
clamps 1-15
cleavage and deprotection 4-3
cytosine content 1-14
definition 1-2
drying 4-2
extinction coefficients 4-11
handling 4-10
homo thymine 1-13
labeling in solution 4-15
labeling support-bound 4-12
labels, effect of 1-12
oligomer length 1-11
orientation 1-10
purification 4-8
purine content 1-11
quantitation 4-11
sequence design 1-10
sequences to avoid 1-13
stability 1-13
strand invasion 1-9, 1-14
structure 1-2
thermal stability 1-7
triplex formation 1-13
PNA synthesis
see also Synthesis
cycle 1-6
monomers 1-3
on Expedite instrument 1-5
overview 1-2
protocol available 1-5
troubleshooting 5-2
waste composition C-8
Post-synthesis procedures
analysis 4-6
cleavage and deprotection 4-3
Q
Quantitation of PNA 4-11
R
Reagents
contents of kit 3-6
flushing requirements 3-8
loading 3-11
location 3-13
ordering B-1
preparing 3-6
resetting volume 3-14
safety information A-1
Related documents x
Index-3
I
N
D
E
X
I
N
D
E
X
S
Safety information A-1
Sequence
design of PNA 1-10
direction 3-5
entering 3-5
structures to avoid 1-13
Software
if PNA not displayed 2-3
installing 2-2
version number 2-2
Spacer, Fmoc-AEEA-OH, see FmocAEEA-OH spacer
Specificity of binding 1-9
Stoichiometry
duplex 1-10
triplex 1-13
Stop button 3-19
Stopping a synthesis 3-19
Strand invasion 1-9, 1-14
Synthesis
see also PNA synthesis
entering sequence 3-5
flushing the system 3-8
interrupting 3-20
key steps 3-2
loading reagents 3-11
monitoring 3-19, 3-20
overview 3-2
post-synthesis procedures 4-2
preparing reagents 3-6
priming 3-8, 3-9, 3-15
running 3-19
selecting chemistry 3-3
starting 3-19
steps in 3-2
Index-4
Applied Biosystems
stopping 3-19
T
Technical documents, obtaining D-9
Technical support, contacting
Africa and the Middle East D-5
Eastern Asia, China, Oceania D-6
e-mail D-2
Europe D-6
Japan D-7
Latin America D-7
telephone or fax in North
America D-3
TFA, safety precautions A-9
Thermal stability of PNA 1-7
Thiol-specific labels 4-17
Triplex formation 1-13
Troubleshooting 5-2
U
Users guide structure x
Users guide structure ix
V
Version number of software 2-2
Volume, resetting reagent 3-14
W
Wash solutions (WshA, WshB), safety
precautions A-10
Waste composition C-8
Waste disposal A-1
World wide web address,Applied
Biosystems xi
Headquarters
850 Lincoln Centre Drive
Foster City, CA 94404 USA
Phone: +1 650.638.5800
Toll Free: +1 800.345.5224
Fax: +1 650.638.5884
Worldwide Sales Offices
Applied Biosystems vast distribution and
service network, composed of highly trained
support and applications personnel, reaches
into 150 countries on six continents. For
international office locations, please call our
local office or refer to our web site at
www.appliedbiosystems.com.
www.appliedbiosystems.com
an Applera business