SeqLab_Exosap_USB.pdf

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ExoSAP-IT

Product Number 78200 100 reactions78201 500 reactions78202 2000 reactions

STORAGEStore at -20 C.

Warning: For research use only. Notrecommended or intended for diagnosis ofdisease in humans or animals. Do not useinternally or externally in humans or animals.


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CONTENTSProduct 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


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PRODUCT DESCRIPTIONExoSAP-ITExonuclease I and Shrimp Alkaline Phosphatase in buffer.

STORAGEStore at -20 C and keep on ice while pipeting. The enzymes are heat labile andwill slowly lose activity at room temperature. Do not store in a frost-free freezer(the temperature rises above 0 C daily).

QUALITY CONTROLAll lots are functionally tested by USB using radiolabeled-dATP and a controlM13 clone single-stranded DNA template as described in the SequenasePCR* Product Sequencing Kit (PN70170) protocol. Release specifications arebased on sequence length, band intensity and sequence quality. The entirePCR product sequence must be readable on a standardized gel with less than24 hours exposure. The sequence must also be free of background bandsstrong enough to interfere with sequence interpretation.

SAFETY WARNINGS AND PRECAUTIONSWarning: For research use only. Not recommended or intended fordiagnosis of disease in humans or animals. Do not use internally orexternally in humans or animals.

Caution: All chemicals should be considered as potentially hazardous. Wetherefore recommend that this product is handled only by those persons whohave been trained in laboratory techniques and that it is used in accordancewith the principles of good laboratory practice. Wear suitable protective clothing,such as lab coats, safety glasses and gloves. Care should be taken to avoidcontact 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.


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OVERVIEWExoSAP-IT prepares PCR products for numerous applications, such assequencing by either radioactive or fluorescent detection methods. When PCRamplification is complete, any unconsumed dNTPs and primers remaining in thePCR product mixture will interfere with these methods. ExoSAP-IT utilizes twohydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, toremove these unwanted dNTPs and primers. The Exonuclease I degradesresidual single-stranded primers and any extraneous single-stranded DNAproduced by the PCR. The Shrimp Alkaline Phosphatase hydrolyzes remainingdNTPs from the PCR mixture which would interfere with the sequencingreaction. ExoSAP-IT is added directly to the PCR product. The enzymes areactive in the buffer used for PCR, hence no buffer exchange is required. Aftertreatment, ExoSAP-IT is inactivated simply by heating to 80 C for 15 minutes.

ExoSAP-IT consists of one pipeting step and two incubations. Just addExoSAP-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 aminimum of hands-on time. Only simple pipette transfers are required,therefore many samples can be processed at once, either manually or withrobotic devices.

After the clean-up procedure is complete the sequencing protocol of choiceshould be followed as described in the specific sequencing kit protocol booklet.

When using automated sequencing reagents, it has been found that PCRproduct clean-up is not always necessary with dye-primer fluorescentsequencing methods, however, in many cases it is beneficial. When sequencingwith fluorescent dye-terminators, it is essential that the PCR product be free ofexcess primers and dNTPs.


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Primer A Primer B4 dNTPs

DNA polymerase,thermal cycling

+

Target DNA

Double-strandedPCR product

4 dNTPsExcess primers

andnucleotides

Add ExoSAP-IT

+ Nucleosides, P i

Double-strandedPCR product

37 C, 15 min for treatment80 C, 15 min to inactivate

Sequencing

Figure 1: Schematic of the ExoSAP-IT method.


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EXOSAP-IT PCR CLEAN-UP PROTOCOLExoSAP-IT may be used to clean-up PCR products ranging in size from lessthan 100bp up to over 20kb. No PCR product will be lost when using theExoSAP-IT clean-up method. This is because the product is not subject to apurification method which may lead to sample loss, such as methods involvingprecipitations or columns.

1. PCR Product*: 5 lExoSAP-IT: 2 l

Total: 7l*Refers to the mixture after PCR amplification is complete.

Note : When treating 10 l or more of PCR product, increase the amount ofExoSAP-IT proportionally.

2. Mix and incubate at 37 C for 15 minutes. (It is convenient to do this step in athermal cycler.)

3. Inactivate ExoSAP-IT by heating to 80 C for 15 minutes. (It is alsoconvenient to do this in a thermal cycler.)

4. The DNA is now ready for direct sequencing using manual or automatedmethods.


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RELATED PRODUCTSProduct Application Pack size Product numberPCR Nucleotide Mixes Functionally tested in 10mM, 500 l 77212

long PCR 25mM, 500 l 77119Sequenase Version For non-cycle 100 70770

2.0 DNA Sequencing Kit radioactive sequencing templatesThermo Sequenase For cycle radioactive 100 78500Cycle Sequencing Kit sequencing templatesThermo Sequenase For cycle radioactive 50 188403 WithRadiolabeled Terminator sequencing templates 33 PddNTPsCycle Sequencing Kit 79750 Without

33 PddNTPs

USB CORPORATION

USACleveland, Ohio800.321.9322www.usbweb.com

USB Authorized Distributor: GE HealthcareAsia PacificTel: +852 2811 8693AustraliaTel: +61 2 9894 5188AustriaTel: 01 57 606 16BelgiumTel: 0800 73888CanadaTel: 1 800 463 5800Central and EastEuropeTel: +43 1 982 3826

DenmarkTel: +45 16 24 00FinlandTel: (09) 512 3940

Former Soviet UnionTel: +7 (095) 232 0250FranceTel: 01-6935-6700GermanyTel: 0761 4903ItalyTel: 02 27322 1JapanTel: 81 3 5331 9317Latin AmericaTel: +55 11 3667 5700Middle East and Africa

Tel: +30 (1) 96 00 687NetherlandsTel: 0165 580 410NorwayTel: 23 18 58 00

PortugalTel: 01 417 70 35South East AsiaTel: 60 3 724 2080South East EuropeTel: +43 (1) 982 3826SpainTel: 935 944 950SwedenTel: 018 16 19 00SwitzerlandTel: 01 802 81 50UK

Tel: 0800 616 928USATel: 1 800 526 3593


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