TaKaRa EpiTaq™ HS - Clontech Laboratories, Inc.
Contents
1. v201402Da 077 543 6116 TaKaRa Be 077 543 19772. 20mM Tris HCI pH8 0 100mM KCI 0 1 mM 1mM 0 5 0 5 50 Tween 20 Nonidet P 40 Glycerol dNTP Mixture 2 5 mM dATP dCTP dGTP dTTP PCR 98 pH7 9 DNA 743 30 10 nmol 1U 25mM TAPS pH9 3 25 50mM KCI 2mM MgCb 0 1mM DTT 200 MM dATP dGTP dCTP 100 uM BEHI dTTP 0 25 mg ml DNA OnE 1 10U 0 6 ug D A Hind Ill 74 1 DNA 2 10U 0 6 g supercoiled pBR322 DNA 74 1 DNA 3 10U 0 6 ug ADNA 74 1 DNA
3. One DNA PCR PCR TaKaRa EpiTaq HS PCR A 1 PCR T Vector dUTP DNA PCR 148 bp 55 C 10 EpiTaq 90 PCR total50 ul TaKaRa EpiTaq HS 5 U wl 0 25 ul 1 25U 50 ul 10 X EpiTaq PCR Buffer Mg2 free 5 ul 1X 25 mM MgClz 5 ul 2 5 mM dNTP Mixture 4 2 5 mM 6 ul 0 3 mM Template lt 100 ng Primer 1 20 pmol 0 4 uM Primer 2 20 pmol 0 4 uM upto 50 ul MgClz gt dNTP
4. Mixture PCR PCR DNA Taq PCR 7gg 98C 10sec 1 55 30sec 72C 30sec 500 bp or 1 min 500 bp 1 kb 2 1 98 5 10 sec 94 C 20 30 sec 2 30 sec 500 bp 1 min 500 bp 1 kb 1 kb 1 min 1 kb 30 40 cycles
5. TaKaRa EpiTaq HS for bisulfite treated DNA Code No R110A Size 250 units Supplied Reagents 10X EpiTaq PCR Buffer Mg2 free dNTP Mixture 2 5 mM each 25 mM MgCl2 Lot No Concentration Volume Expiration Date Shipping at 20 C Store at 20 C 5 units ul 50 ul For a detailed protocol refer to the product user manual Storage Buffer 20mM Tris HCI pH8 0 100mM KCl 0 1mM EDTA 1mM DTT 0 5 Tween 20 0 5 NonidetP 40 50 Glycerol 1ml 1 2 ml 1 2 ml Supplied dNTP Mixture 2 5 mM each dNTP Mixture is ready for use in PCR without dilution Form Dissolved in water sodium salts pH7 9 Purity 98 for each dNTP Unit definition One unit is the amount of enzyme that will incorporate 10 nmol of dNTPs into acid insoluble products in 30 min at 74 C with activated salmon sperm DNA as the template primer Reaction mixture for unit definition 25mM TAPS pH9 3 at 25 C 50mM KCl 2mM MgCl 0 1mM DIT 200 uM eachdATP dGTP dCTP 100 uM BEHI dTTP 0 25 mg ml activated salmon sperm DNA Purity Nicking endonuclease and exonuclease activity were not de tected after incubation of 0 6 u g of supercoiled pBR322 DNA 0 6 u g of A DNA or 0 6 u gof A Hind Ill digest with 10 units of this enzyme for 1 hour at 74 C Application PCR amplification using bisulfite treated DNA as a template PCR products As most PCR products amplified with TaKaRa EpiTaq HS have one Aat the 3
6. alifornia 94404 USA EpiTaq is a trademark of TAKARA BIO INC Note This product is for research use only It is not intended for use in therapeutic or diagnostic procedures for humans or animals Also do not use this product as food cosmetic or household item etc Takara products may not be resold or transferred modified for resale or transfer or used to manufacture commercial products without written approval from TAKARA BIO INC If you require licenses for other use please contact us by phone at 81 77 543 7247 or from our website at www takara bio com Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page It is your responsibility to review understand and adhere to any restrictions imposed by such statements All trademarks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions v201402Da TaKaRa EpiTaq HS for bisulfite treated DNA etic ae Shipping at 20 C JERS HEIS Storeat 20 C 10 X EpiTaq PCR Buffer Mg free 1ml dNTP Mixture 2 5 mM 1 2 ml 25 mM MgCl2 1 2 ml Lot No
7. termini the obtained PCR products can be directly cloned into T vectors Also it is possible to clone the products in blunt end vectors after blunting and phosphorylation of the ends PCR test Good performance by PCR was confirmed by amplification of a 148 bp fragment from a DNA template containing dUTP Antibody mediated inhibition of EpiTaq polymerase activity was con firmed to be more than 90 after the reaction was incubated at 55 C for 10 min General reaction mixture for PCR total 50 pl Volume Final conc TaKaRa EpiTaq HS 5 U u1 0 25 ul 1 25U 50 ul 10X EpiTaq PCR Buffer Mg free 5 ul 1X 25 mM MgCl2 5 ul 2 5 mM dNTP Mixture 2 5 mM each 6 ul 0 3mM Template lt 100 ng Primer 1 20 pmol 0 4 uM Primer 2 20 pmol 0 4 uM Sterile distilled water up to 50 ul Try the above reaction mixture first If necessary change the concentration of MgCl2 dNTP Mixture or primers to improve amplification efficiency extension or specificity Please refer to the Troubleshooting section in the product user manual for additional details PCR conditions This enzyme can be used with standard PCR conditions since the monoclonal antibody is denatured in the initial DNA denaturation step There is no need for an additional step to denature the antibody to Taq polymerase Example Amplification of a 0 5 kb DNA fragment 98 10sec 55 30sec 30 40 cycles 72 C 30sec for lt 500 bp or 1 min for 500 bp 1 kb lt 1 Denat
8. uration conditions vary depending on the thermal cycler and tubes used for PCR Denaturation for 5 10 sec at 98 C or 20 30sec at 94 C is recommended 2 Please set it according to the amplicon size The standard setting is 30 sec for products lt 500 bp and 1 min for products 500 bp 1 kb in size For amplification of targets longer than 1 kb the standard setting is 1 min kb NOTICE TO PURCHASER LIMITED LICENSE P1 PCR Notice Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 789 224 5 618 711 6 127 155 and claims outside the US corresponding to expired US Patent No 5 079 352 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim no right to perform any patented method and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosys tems 850 Lincoln Centre Drive Foster City C
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