E.coli Alkaline Phosphatase 取扱説明書
Contents
1. TEL 06 6348 3786 FAX 06 6348 3833 E mail order_lifescience toyobo jp TEL 03 6887 8819 FAX 03 6887 8951 E mail order_lifescience toyobo jp TEL 06 6348 3888 9 00 12 00 13 00 17 00 E mail tech_osaka toyobo jp URL http Awww toyobo co jp bio Code No BAP 111 14 04 E coli Alkaline Phosphatase Manual 1 Composition of Supplied Reagent 10 x Reaction Buffer for BAP 500mM Tris HCI pH 8 0 10mM MgClz 2 Dephosphorylation of DNA fragments by BAP Sterilized disitilled water 175 X wl DNA fragment X ul 5 termini 5 20 pmoles Reaction Buffer 20 ul BAP 0 1 1U 1 5 ul 2U Total 200 ul Incubate at 60 C 3 protruding end blunt end or at 37 C 5 protruding end for 1hr Add 200 of TE saturated phenol pH8 0 Vortex Centrifuge at 12 000 rpm for 1 min Transfer upper layer to a new tube Add 500 gl of chloroform Vortex Centrifuge at 12 000 rpm for 1 min Repeat this step 3 times Transfer upper layer to a new tube Add 20 u I of 3M Na Acetate pH5 2 Add 500 gl of cold EtOH Vortex Incubate at 70 C for 5 min Centrifuge at 15 000 rpm for 10 min Remove the supernatant carefully f Rinse with 1ml of 70 EtOH Dry under vacuum for 5 min XBecause B2. AP is very stable and hard to be inactivated by heating it is recommended to purify DNA fragments by Phenol Chloroform treatment after the dephosphorylation reaction In stead of Phenol Chloroform treatment DNA fragment purification Kit Mag Extractor PCR amp Gel Clean up Code NPK 601 TOYOBO is also available TOYOBO CO LTD Life Science Department 2 8 Dojima hama 2 Chome Kita ku Osaka 530 8230 JAPAN URL http www toyobo co jp e bio
3. Code No BAP 111 14 04 E coli Alkaline Phosphatase 1 10 x Reaction Buffer for BAP 500mM Tris HCI pH 8 0 10mM MgCle 2 BAP 175 X ul DNA fragment X 5 5 20 pmoles Reaction Buffer 20 ul BAP 0 1 1U 1 5 ul 2U Total 200 ul 60 3 37 C 5 1hr M 12 000 rpm x 1 min 500 gl 12 000 rpm x 1 min TE pH8 0 200 gl 3 3M pH5 2 2041 500 ul 70C 5min 15 000 rpm x 10 min DNAGRERAIEL TT 70 1ml 5min BAP DNA DNA MagExtractor PCR amp Gel Clean up Code NPK 601 TOYOBO
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