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Application Note 2013〈16〉 Customer feedback on products Product Name Manufacturer Application : KAPA MG Kit (KK7150MG) KAPATaqEXtra HotStart Readymix with Dye (KK 3606) : KAPA BIOSYSTEMS : Genotyping of knock-out mice The following data were provided by the courtesy of Dr. Mamoru Aoto of Department of Circulatory Physiology, Graduate School of Medicine, Ehime University, Japan. Experimental conditions ①Conventional method (Manufacturer T's product) <Extraction of mouse tail DNA> Mouse tail(2mm) in 1.5ml tube ↓ + 75µl Alkaline Lysis Buffer (25mM NaOH, 0.2mM EDTA) ↓ 95℃, 30min ↓ 4℃ ↓ + 75µl Neutralization Buffer (40mM TrisHCl, pH 5) Vortex well <PCR reaction mixture composition> 10× Reaction buffer 2.5µl 2µl 2.5mM MgCl2 2.5mM dNTP mix 2.5µl 10µM Fwd Primer 1.25µl 10µM Rev Primer 1.25µl Template 1µl Manufacturer T's enzyme 0.25µl MilliQ water 14.25µl total 25µl ②KAPA MG Kit <Extraction of mouse tail DNA> Followed the instructions attached to the kit. ③KAPATaq EXtra HotStart ReadyMix with dye (2×) <Extraction of mouse tail DNA> Same as the conventional method. <PCR reaction mixture composition> KAPA2G Robust HotStart ReadyMix (2x) 12.5 µl 10µM Fwd Primer 1.25µl 10µM Rev Primer 1.25µl Template 1µl MilliQ water 9µl total 25µl <PCR reaction mixture composition> KAPATaq EXtra HotStart ReadyMix with dye (2x) 12.5 µl 10µM Fwd Primer 1.25µl 10µM Rev Primer 1.25µl Template 1µl MilliQ water 9µl total 25µl ● PCR device:BIO-RAD T100 Thermal Cycler ● PCR program 95 ℃, 3min ×1 cycle 95 ℃, 15sec 64 ℃, 15sec ×35 cycle 72 ℃, 15sec 16 ℃, ∞ ● Target amplicon size:163bp Results KAPA Conventional method MG Kit Manufacturer T ++−−++−− KAPATaq Conventional method EXtra Manufacturer T ++−−++−− The operation procedures and PCR results were compared among two KAPA kits (KAPA MG Kit, KAPA TaqEXtra HotStart ReadyMix with dye) and a conventional method (Manufacturer T). Summary of comparison between KAPA kits and the conventional method (Manufacturer T) ・Neither of the kits detected any non-specific bands that were detected in the conventional method. ・Both kits were equivalent to the conventional method in terms of amplification efficiency. ・Both kits support easy preparation of the PCR reaction mixture and therefore prevent mistakes (particularly for beginners). ・KAPA MG Kit requires shorter time for DNA extraction compared to the conventional method. ・KAPA TaqEXtra contains a dye, so electrophoresis can be performed directly after the PCR. M : Marker 1, 2 : Knock-out mice (+) 3, 4 : Wild-type mice (−) Electrophoresis conditions 1% Agarose gel, 100V, 30 min Reaction mixtures were applied in 10 μl aliquots. The marker was applied in 5 μl aliquot. <Customer's comments> We have been detecting KO alleles by PCR for genotyping. Both of the Kapa Biosystems kits suppressed non-specific amplification and produced easy-to-comprehend results. Inexperienced students (medical school freshmen) could successfully perform genotyping without making any mistakes. Nippon Genetics Co.,Ltd http://www.n-genetics.com Head Office : Koraku Mori Bldg.18F, 4-14 Koraku 1-Chome, Bunkyo-ku, Tokyo, 112-0004, Japan West Sales Office : Raffine Oike 3F, 565, Nishinotoin Oike sagaru, Nakagyo-ku, Kyoto, 604-8277 Japan Nippon Genetics Europe GmbH : Binsfelder Strasse 77, 52351 Dueren Germany TEL : +81-(0)3-3813-0961 FAX : +81-(0)3-3813-0962 TEL : +81-(0)75-257-5421 FAX : +81-(0)75-257-5422 KP2013AUG7150MG