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 stock pAMD241 (14028s dCas3::thyA  + Salmonella CRISPRi library).  Mix gently.  Add 250 uL each glycerol stock to 12.5 mL LB+ 100ug/mL ampicillin. Measure OD600.  Dilute again 1 mL culture in 12.5 mL LB+/mL ampicillin,  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.     For experiment, thaw 3 aliquots frozen cells. Wash 2x in water, resuspend in 190 ul LB + 100mM MOPS + 100ug/mL ampicillin. Add 18.7 ul cells to 4 mL LB + 100mM MOPS + 100ug/mL ampicillin and add to 4mL LB + 100mM MOPS + 100ug/mL ampicillin + 3% bile salts.  Grow 4.5 hours at 37C/225 rpm.  Miniprep cells for “set1”. Dilute to OD600=0.01 (4 mL volume).  Grow 4.5 hours at 37C/225 rpm. Miniprep cells for “set2”. Miniprep remaining cells form frozen aliquot for “start”   Cells are resuspended in LB + 100mM  MOPS + 100ug/mL ampicillin, with or without 3% bile salt treatment. (sodium choleate, SIGMA S9875)  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.