ÐÏࡱá > þÿ . 0 þÿÿÿ - ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿì¥Á %` ð¿ å bjbj"x"x . @ @ × ÿÿ ÿÿ ÿÿ ¤ Z Z Z Z Z Z Z n v v v 8 ®  n » ì ê ê ê ê ê ê ê ê : < < < < < < $ § h f ` Z ê ê ê ê ê ` Z Z ê ê u ú ú ú ê H Z ê Z ê : ú ê : ú ú : î , Z Z v ê Þ 5É÷K4É v 2 p : ‹ 0 » $ R u ¢ u v u Z v Ä ê ê ú ê ê ê ê ê ` ` ¢ X ê ê ê » ê ê ê ê n n n ä R $ n n n R n n n Z Z Z Z Z Z ÿÿÿÿ BL21 Competent Cells Preparation: Prepare an overnight streak plate of BL21 Comp Cells grown on an LB/KAN plate. Take Kanamycin out of freezer to thaw. Thoroughly clean (and bleach if necessary) 2 liter growth bottles and centrifuge bottles to be used for BL21 preparation. Label 1.5mL Eppendorf tubes and 15mL blue cap conical tubes with “BL-21” and today’s date. Place these in the freezer. Place two disposable 25mL pipets in the freezer. Place centrifuge bottles in freezer. Procedure: In each growth bottle add 500mL of LB, 500ul of kanamycin, 10mM of MgSO4, and 0.4% Glucose [1:1 ratio for all]. Using a 25ml pipet, draw 10 – 15mls of the LB/MgSO4/Glucose media out of one of the growth bottles to resuspend/transfer the BL21 cells from the plate. Return resuspended cells to each growth bottle. Swirl gently to mix. Place aluminum foil over the top or tape caps on the bottle. Growth bottles should be incubated at 370C for 4-6 hours at 260-280 rpm. When the OD600 has reached between 0.6 and 0.8, the culture is ready. CELLS AND ALL EQUIPMENT MUST BE KEPT COLD FOR ENTIRE PROCEDURE. Fill two ice buckets with ice for the rest of the procedure. Place wash buffer, competent buffer and dilution buffer from Zymo Kits on ice. Turn centrifuge on and be sure temperature is set to 40C. Culture can be left in the growth bottles or transferred to the centrifuge bottles and placed on ice for at least 10 minutes. If you have not already, transfer the culture to the centrifuge bottles. Centrifuge at 3,000 rpm for 20 minutes. Carefully pour off supernatant and place bottles on ice. If necessary the pellet can be stored at -200C until ready to be processed. Add 30ml of wash buffer and 30ml of dilution buffer to each bottle. Resuspend the cells by swirling buffer around bottle. If necessary, pipet buffer up and down to assist with resuspension. Be sure there are no clumps. Leave bottles sit on ice for 10 minutes. Prepare a container of dry ice. Centrifuge at 3,000rpm for 20 minutes. Carefully pour off supernatant and place bottles on ice. Add 30ml of competent buffer and 30ml of dilution buffer to each bottle. Resuspend cells by swirling buffer around bottle. If necessary, pipet buffer up and down to assist with resuspension. Be sure there are no clumps. Pipet aliquots of competent cells into 1.5ml Eppendorf tubes and 15ml blue cap vials. Keep tubes on ice while proceeding. Place competent cells in liquid nitrogen freezer or -700C. Cells are ready for efficiency testing. # H a d p q ™ 8 O ¼ á 5 6 A ^ _ ‘ ’ “ › > £ ¤ Ï Ò J K L ‰ Š ” • û ü ' øíâÞÚÞÚÞÚÞÖÞÖÞÒÞÊÞÆÞº³ÂÞ«ÞÊÞҝ’‡ÒÒÞÒ{ÒÞÒ{Òw hí4¢ h/(Y hØE h:0 H*h:0 h:0 CJ aJ h:0 56CJ aJ h:0 h:0 56CJ aJ hØE hØE H*h‚TÖ hÒqÛ hØE hÒqÛ H*hÒqÛ hÇ hØE hØE H*h:0 hD h}W› hØE hØE høF¯ CJ, aJ, hØE hØE CJ, aJ, hâ CJ, aJ, . # r ™ ‹ ¼ á â ã î _ > { Ä K L Ù ÷ õ õ í í å í í í à à à Ø Ð È È È ¿ ¿ ¿ È È „h^„hgd:0 &

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