title: 14028s CRISPRi Bile salts
design: 14028s CRISPRi Bile salts
library_construction_protocol: Sample collection and nucleic acid purification happen in the same step. After setting up initial culture, miniprep remaining cells from thawed aliquot (= \"start\").  Use Qiagen Spin Miniprep Kit -include Buffer PB wash, elute in 30 uL water.  After 4.5 hrs growth and dilute to OD600=0.01, take 1.2 mL of culture and miniprep (same miniprep procedure) (= \"set 1\").  After 4.5 hrs growth, take 1.2 mL of culture and miniprep (same miniprep protocol) (= \"set 2\").  Qubit quantify all minipreps. Start and set 2 sample used for this experiment. Thaw glycerol stocks pAMD240 and pAMD241.  Mix gently.  Add 250 uL each glycerol stock to 12.5 mL LB+amp.  Measure OD600.  Dilute again 1 mL culture in 12.5 mL LB+amp.  Record OD600.  Grow o/n at 37deg/225 rpm.  Next day set up 200 uL aliquots of cells + 50 uL 50% glycerol and flash freeze on dry ice.  Thaw aliquot of frozen cells from \"14028s CRISPRi culture setup\" protocol.  Dilute cells to OD600=0.01 (4 mL volume).  Grow 4.5 hours at 37deg/225 rpm.  Dilute to OD600=0.01 (4 mL volume).  Grow 4.5 hours at 37deg/225 rpm. no treatment After setting up initial culture, miniprep remaining cells from thawed aliquot (= \"start\").  Use Qiagen Spin Miniprep Kit -include Buffer PB wash, elute in 30 uL water.  After 4.5 hrs growth and dilute to OD600=0.01, take 1.2 mL of culture and miniprep (same miniprep procedure) (= \"set 1\").  After 4.5 hrs growth, take 1.2 mL of culture and miniprep (same miniprep protocol) (= \"set 2\").  Qubit quantify all minipreps. Start and set 2 sample used for this experiment. use 20 ng of miniprepped DNA (from \"miniprep\" sample extraction protocol) as template for PCR.  Set up 4 x 50 uL PCR for each sample using Taq. Final concentration ultramers = 0.1 uM.  Cycling:  initial denaturation 95deg - 5 min; 10 cycles of 95deg-30 sec, 53deg-40sec, 68deg-1min; final extension 68deg-5 min; 4deg hold.  Combine like PCRs and run 15 uL on acrylamide gel, 110V, 75min.  Divide remaining PCR sample into 2 tubes and AMPure 0.8x clean.  Elute each in 30 uL water, combine like samples, AMPure 0.8x clean again.  Elute 11 uL. Qubit quantify.