Bp clonase manual

BP Clonase® II Enzyme Mix The BP Clonase® II enzyme mix (Box 2) contains the followin g rea ents. Store Box 2 at –20°C for up to 6 months. For long-term storage, store at –80°C. Item Composition Quantity BP Clonase® II Enzyme Mix Proprietary 40 μl Proteinase K solution 2 μg/μl in: 10 mM Tris-HCl, pH 20 mM CaCl 2 50% glycerol 40 μl. BP Clonase™ II enzyme mix catalyzes in vitro recombination between an attB-PCR product (or attB-containing expression clone) and an P-containing donor vector to generate an attL-containing entry clone. Store Gateway® BP Clonase™ II enzyme mix at ºC (non-frost-free freezer) for up to 6 months. For long-term storage, store at ºC. In a ml microcentrifuge tube, prepare the following 15 µl BP reaction: attB DNA ( ng) – µl attP DNA (Invitrogen pDONR vector, ng/µl) µl BP Clonase II enzyme mix µl TE Buffer, pH add to a final volume of 15 µl; Mix well by vortexing briefly and incubate at 25°C for 4 hours.

Generating an Entry clone using a Gateway BP reaction. (A) A Gateway BP reaction uses BP clonase enzymes to recombine an attP site with an attB site to generate an attL site and an attR site, whereas LR clonase performs the reverse recombination.(B) attP, attL, and attR sites have recognition "arms" on either/both sides of the "recognition region" (box with arrowhead), whereas attB. 3 ページ 手順 BP 反応 BP クロナーゼ™II 酵素ミックスは5 倍濃度の溶液です。反応容量を変更する場合は、BP クロナー ゼ™II 酵素ミックスの最終濃度が1 倍となるようにしてください。ポジティブコントロールについ ては、pEXP7-tet を ng（2 μL）ご利用ください。. The attB × attP reaction is mediated by Gatewayfi BP Clonase enzyme mix; the attL × attR reaction is mediated by Gatewayfi LR Clonase enzyme mix. ccdB is the F plasmid-encoded gene that inhibits growth of E. coli (2,3) and figenefl represents any DNA segment of interest (e.g. PCR product, cDNA, genomic DNA). Description.

Gatewayfi BP Clonase enzyme mix is a proprietary enzyme formulation containing the bacteriophage lambda recombination protein Integrase (Int) and the E. coli - encoded protein Integration Host Factor (IHF) (1). Gateway BP Clonase II enzyme mix catalyzes the in vitro recombination of PCR products or subcloning DNA segments from clones (containing attB sites) and a donor vector (containing attP sites) to generate entry clones. Technology with Clonase® II manual for a one-tube protocol. Although this protocol allows you to generate expression clones more rapidly than the standard BP reaction followed by the LR reaction, fewer expression clones will be obtained (generally 10–20% of the total number of entry clones).