Shlo/notebook/TopoClone Topocloning Protocol

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Obtain one tube of TOPO-TA cloning vector. Each tube contains 0.5 ul of cloning vector. Place one 500 ul tube on ice to chill for the transformation step. Briefly spin the tube to collect the TOPO vector at the bottom of the tube. Add 2 ul of your PCR product to the tube. Incubate at room temperature for EXACTLY 5 minutes. Do not allow the ligation reaction to proceed for more than 5 minutes. Transfer the ligation tube to ice. Aliquot 25 ul of competent cells to your chilled tube. (Each aliquot of cells is enough for two reactions). To each aliquot of cells, add 1 ul of your ligation mixture. Incubate cells and DNA on ice for 30 minutes. (Use this time to purify your remaining PCR product!) After 25 minutes, bring your ice bucket over to the heatblock. Heat shock your bacteria + DNA for EXACTLY 30 seconds – no more, no less! Immediately place your bacteria back on ice. Add 125 of SOC medium to your cells. Cap the tubes tightly, and bring them to the 37 degree shaking incubator at the back of the room. Tape your tube flat on the shaking platform to allow the tube to shake in a horizontal position. Incubate on the shaker for 25 minutes. Meanwhile, obtain and label a prewarmed LB+amp agar plate. After your 25 minute incubation, use a pipet to gently transfer the entire 125 ul transformation onto your labeled plate. Empty a vial of glass beads onto the agar, place the top on the plate, and spread the cells by rotating and rocking the plate gently (TF will demonstrate). Empty the glass beads into the biohazard trash, and cover the petri plate. Place your plate agar-side up in the 37º incubator.