Berglund:KCM competent

PROTOCOL FOR MAKING KCM COMPETENT CELLS wear gloves, change gloves frequently to reduce contamination 1	Streak competent cells onto an LB plate, grow overnight at 37°C 2	Choose a colony with a toothpick and innoculate 500ml of LB	grow overnight at 37°C with shaking at 215rpm 3	Dilute 1:100 in 500ml ofprewarmed LB, grow to OD 0.4-0.5 (between 2 and 3 hrs), 37°C at 215rpm 4	Spin down in four 250ml prechilled bottles at 5000rpm in the JA-14 for 5 min	put bottles on ice, go to cold room DO THE REST IN THE COLD ROOM Have in the cold room: 25ml pipettes in metal box for cell resuspension Electric pipettor a BIG beaker of 1.5ml eppendorf tubes that are sterile 1ml pipette man and box of 1ml tips TSS solution styrofoam ice bucket with 500ml of 100% ethanol in it + dry ice floaty racks fat sharpie markers with the appropriate color for your competent cells (TG1= green, Novablue=blue, BL21 (DE3) pLyseS= red) paper towels gloves cart (you will work entirely on the cart because there isn't any extra	bench space in the cold room) freezer boxes labelled appropriately (name of cells in the appropriate color	that matches the color on the tops of their lids) sterile spatula 3 eppendorf tube racks TSS solution (makes100ml of solution): 10g PEG 3350 ( fc=10%) 5ml DMSO (fc=5%) 2ml 1M MgCl2 (fc=20mM) LB broth to bring the volume up to 100ml pH should be 6.5, check using paper and sterile tips 5	Scoop pellets using spatula into one centrifuge bottle 6	Add 25-50ml of the ICE COLD TSS to the combined pellets 7	Resuspend pellets using the prechilled pipettes and the electric pipettor try hard to not get the cell suspension into the pipettor, homogenize well 8	place 20 tubes or so into a rack 9	add 25-50ml of the ICE COLD TSS to the combined pellets 10	Close the lids, take the sharpie with the right color and mark a line across every lid to indicate the what cell it is 11	place tubes into floaty rack, place rack into -80° ethanol/dry ice mix wait until mix has stopped boiling 12	take tubes out, place into freezer box, place box on ice 13	repeat until all of the cell suspension has been aliquoted out 14	run to our -80° freezer and put the freezer box inside (you don't want the	cells to thaw once frozen, you must work very quickly) Clean up, go home. You've worked hard :)