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ExoSAP-IT

Product Number 78200 100 reactions


78201 500 reactions
78202 2000 reactions
STORAGE
Store at -20C.
Warning: For research use only. Not
recommended or intended for diagnosis of
disease in humans or animals. Do not use
internally or externally in humans or animals.
2
CONTENTS
Product Description ........................................................................................... 3
Storage................................................................................................................. 3
Quality Control .................................................................................................... 3
Safety Warnings and Precautions ................................................................ 3, 8
Overview.............................................................................................................. 4
ExoSAP-IT PCR Clean-up Protocol .................................................................. 6
Related Products ................................................................................................ 7
Contact Information ........................................................................................... 7
Patent and License Information...................................................................... 12
3
PRODUCT DESCRIPTION
ExoSAP-IT
Exonuclease I and Shrimp Alkaline Phosphatase in buffer.
STORAGE
Store at -20C and keep on ice while pipeting. The enzymes are heat labile and
will slowly lose activity at room temperature. Do not store in a frost-free freezer
(the temperature rises above 0C daily).
QUALITY CONTROL
All lots are functionally tested by USB

using radiolabeled-dATP and a control


M13 clone single-stranded DNA template as described in the Sequenase
PCR* Product Sequencing Kit (PN70170) protocol. Release specifications are
based on sequence length, band intensity and sequence quality. The entire
PCR product sequence must be readable on a standardized gel with less than
24 hours exposure. The sequence must also be free of background bands
strong enough to interfere with sequence interpretation.
SAFETY WARNINGS AND PRECAUTIONS
Warning: For research use only. Not recommended or intended for
diagnosis of disease in humans or animals. Do not use internally or
externally in humans or animals.
Caution: All chemicals should be considered as potentially hazardous. We
therefore recommend that this product is handled only by those persons who
have been trained in laboratory techniques and that it is used in accordance
with the principles of good laboratory practice. Wear suitable protective clothing,
such as lab coats, safety glasses and gloves. Care should be taken to avoid
contact with skin or eyes. In the case of contact with skin or eyes wash
immediately with water.
See MSDS (Material Safety Data Sheet) on p. 8.
4
OVERVIEW
ExoSAP-IT

prepares PCR products for numerous applications, such as


sequencing by either radioactive or fluorescent detection methods. When PCR
amplification is complete, any unconsumed dNTPs and primers remaining in the
PCR product mixture will interfere with these methods. ExoSAP-IT utilizes two
hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, to
remove these unwanted dNTPs and primers. The Exonuclease I degrades
residual single-stranded primers and any extraneous single-stranded DNA
produced by the PCR. The Shrimp Alkaline Phosphatase hydrolyzes remaining
dNTPs from the PCR mixture which would interfere with the sequencing
reaction. ExoSAP-IT is added directly to the PCR product. The enzymes are
active in the buffer used for PCR, hence no buffer exchange is required. After
treatment, ExoSAP-IT is inactivated simply by heating to 80C for 15 minutes.
ExoSAP-IT consists of one pipeting step and two incubations. Just add
ExoSAP-IT to the PCR product and within 30 minutes be ready to sequence
(Figure 1). All gel or column purifications, sedimentations, filtrations, beads and/
or magnetic separations are eliminated. The method is designed to require a
minimum of hands-on time. Only simple pipette transfers are required,
therefore many samples can be processed at once, either manually or with
robotic devices.
After the clean-up procedure is complete the sequencing protocol of choice
should be followed as described in the specific sequencing kit protocol booklet.
When using automated sequencing reagents, it has been found that PCR
product clean-up is not always necessary with dye-primer fluorescent
sequencing methods, however, in many cases it is beneficial. When sequencing
with fluorescent dye-terminators, it is essential that the PCR product be free of
excess primers and dNTPs.
5
Primer A Primer B 4 dNTPs
DNA polymerase,
thermal cycling
+
Target DNA
Double-stranded
PCR product
4 dNTPs
Excess primers
and
nucleotides
Add ExoSAP-IT
+
Nucleosides, P
i
Double-stranded
PCR product
37C, 15 min for treatment
80C, 15 min to inactivate
Sequencing
Figure 1: Schematic of the ExoSAP-IT method.
6
EXOSAP-IT PCR CLEAN-UP PROTOCOL
ExoSAP-IT may be used to clean-up PCR products ranging in size from less
than 100bp up to over 20kb. No PCR product will be lost when using the
ExoSAP-IT clean-up method. This is because the product is not subject to a
purification method which may lead to sample loss, such as methods involving
precipitations or columns.
1. PCR Product*: 5l
ExoSAP-IT: 2l

Total: 7l
*Refers to the mixture after PCR amplification is complete.
Note: When treating 10l or more of PCR product, increase the amount of
ExoSAP-IT proportionally.
2. Mix and incubate at 37C for 15 minutes. (It is convenient to do this step in a
thermal cycler.)
3. Inactivate ExoSAP-IT by heating to 80C for 15 minutes. (It is also
convenient to do this in a thermal cycler.)
4. The DNA is now ready for direct sequencing using manual or automated
methods.
7
RELATED PRODUCTS
Product Application Pack size Product number
PCR Nucleotide Mixes Functionally tested in 10mM, 500l 77212
long PCR 25mM, 500l 77119
Sequenase Version For non-cycle 100 70770
2.0 DNA Sequencing Kit radioactive sequencing templates
Thermo Sequenase For cycle radioactive 100 78500
Cycle Sequencing Kit sequencing templates
Thermo Sequenase For cycle radioactive 50 188403 With
Radiolabeled Terminator sequencing templates
33
PddNTPs
Cycle Sequencing Kit 79750 Without
33
PddNTPs
USB CORPORATION
USA
Cleveland, Ohio
800.321.9322
www.usbweb.com
USB Authorized Distributor: GE Healthcare
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8
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11

Exonuclease I/Shrimp Alkaline Phosphatase method of use covered by one or more of


the following US patents: 5,756,285 and 5,741,676. ExoSAP-IT patent pending.
Sequenase is a trademark of GE Healthcare Bio-Sciences Ltd or its subsidiaries.
*The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche
Molecular Systems and F. Hoffmann-La Roche Ltd.
ExoSAP-IT is a trademark of USB Corporation.
USB and logo design are registered trademarks of USB Corporation.
USB Corporation 2000 - All rights reserved
Printed in the United States
USB Corporation
26111 Miles Road
Cleveland, Ohio 44128
www.usbweb.com 00-78200A/00/06