{"text_format":"1","workbook":"xlsx","n_sheets":4,"sheets":["Sect 1 Growth & Thio-labelling","Sect 2 RNA Prep","Sect 3 Biotinylation ","Sect 4 Beads"],"not_rendered":{"charts":0,"images":0,"macros":0,"external_links":2,"defined_names":2,"comments":0,"pivot_tables":0},"unopened":[],"bounds":{"document_bytes":16777216,"sheet_cells":100000,"rows_first":2000,"rows_last":500,"rows_between":2000,"halvings":0,"cell_chars":16384,"cell_head_chars":4096},"cells_cut":0,"cells_raw":0,"read":"whole","date_system":"1900"}

{"sheet":"Sect 1 Growth & Thio-labelling","index":0,"n_rows":33,"n_cols":19,"rows_kept":33,"cut":false,"hidden":false,"merged":[[0,6,0,8],[1,6,1,8],[2,6,2,8],[3,6,3,8],[13,6,13,8],[3,1,3,2],[3,3,3,4],[12,9,12,11],[4,6,4,8],[10,6,10,8],[11,6,11,8],[12,6,12,8],[9,6,9,8],[9,9,9,11],[7,6,7,8],[7,9,7,11],[5,0,5,2],[10,9,10,11],[11,9,11,11],[0,9,0,11],[1,9,1,11],[2,9,2,11],[3,9,3,11],[4,9,4,11],[0,15,0,17],[0,12,0,14],[1,12,1,14],[1,15,1,17],[2,12,2,14],[2,15,2,17],[1,0,2,0],[1,1,2,4],[14,12,14,14],[14,15,14,17],[15,12,15,14],[15,15,15,17],[3,12,3,14],[3,15,3,17],[4,12,4,14],[4,15,4,17],[10,12,10,14],[10,15,10,17],[13,9,13,11],[14,9,14,11],[15,9,15,11],[15,6,15,8],[17,18,32,18],[17,7,17,8],[17,10,17,11],[17,13,17,14],[17,16,17,17],[29,1,31,4],[16,12,16,14],[16,15,16,17],[11,12,11,14],[11,15,11,17],[12,12,12,14],[12,15,12,17],[13,12,13,14],[13,15,13,17],[16,6,16,8],[14,6,14,8],[16,9,16,11],[0,0,0,5],[17,5,18,5],[5,18,6,18],[5,15,6,17],[5,12,6,14],[5,9,6,11],[5,6,6,8],[6,0,6,5],[7,12,7,14],[7,15,7,17],[8,6,8,8],[8,9,8,11],[8,12,8,14],[8,15,8,17],[9,12,9,14],[9,15,9,17]]}
["1. Growth and Thiolation",null,null,null,null,null,"Culture 1",null,null,"Culture 2",null,null,"Culture 3",null,null,"Culture 4"]
["Date",{"$f":"TODAY()","v":"2019-05-14"},null,null,null,"Name"]
[null,null,null,null,null,"1.3 OD600"]
[null,"Stock Conc",null,"Final Conc",null,"Time"]
["Culture volume",null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,"mL"]
["1.5 S. pombe spike",null,null,"enter in this row →",null,null,null,null,null,null,null,null,null,null,null,null,null,null,"µL"]
["1 aliquot = 400 mL of culture"]
["1.6 β-est",10,"mM",10,"µM",{"$f":"B8/(D8/1000)","v":1000},{"$f":"(G5/$F8)*1000","v":0},null,null,{"$f":"(J5/$F8)*1000","v":0},null,null,{"$f":"(M5/$F8)*1000","v":0},null,null,{"$f":"(P5/$F8)*1000","v":0},null,null,"µL"]
["Incubation time",null,null,null,null,"For",null,null,null,null,null,null,null,null,null,null,null,null,"min"]
["Sample taken out?",null,null,null,null,"Vol",null,null,null,null,null,null,null,null,null,null,null,null,"mL"]
["1.6 IAA",1.5,"M",0.75,"mM",{"$f":"B11/(D11/1000)","v":2000},{"$f":"((G5-G10)/$F11)*1000","v":0},null,null,{"$f":"((J5-J10)/$F11)*1000","v":0},null,null,{"$f":"((M5-M10)/$F11)*1000","v":0},null,null,{"$f":"((P5-P10)/$F11)*1000","v":0},null,null,"µL"]
["Incubation time",null,null,null,null,"For",null,null,null,null,null,null,null,null,null,null,null,null,"min"]
["Sample taken out?",null,null,null,null,"Vol",null,null,null,null,null,null,null,null,null,0,null,null,"mL"]
["1.7 4tU",100,"mM",10,"µM",{"$f":"B14/(D14/1000)","v":10000},{"$f":"((G5-(G10+G13))/$F14)*1000","v":0},null,null,{"$f":"((J5-(J10+J13))/$F14)*1000","v":0},null,null,{"$f":"((M5-(M10+M13))/$F14)*1000","v":0},null,null,{"$f":"((P5-(P10+P13))/$F14)*1000","v":0},null,null,"µL"]
["Incubation time",null,null,null,null,"For",null,null,null,null,null,null,null,null,null,null,null,null,"sec"]
["Sample taken out?",null,null,null,null,"Vol",null,null,null,null,null,null,null,null,null,0,null,null,"mL"]
["1.7 Uridine",1,"M",5,"mM",{"$f":"B17/(D17/1000)","v":200},{"$f":"((G5-(G16+G13+G10))/$F17)","v":0},null,null,{"$f":"((J5-(J16+J13+J10))/$F17)","v":0},null,null,{"$f":"((M5-(M16+M13+M10))/$F17)","v":0},null,null,{"$f":"((P5-(P16+P13+P10))/$F17)","v":0},null,null,"mL"]
["Time Course",null,null,null,null,"Sample #","Time","Vol (mL)",null,"Time","Vol (mL)",null,"Time","Vol (mL)",null,"Time","Vol (mL)",null,"Time Minutes (min) or Seconds (s)   - Vol in mL"]
[null,null,null,null,null,null,"s or min","Cult ure","Me OH","s or min","Cult ure","Me OH","s or min","Cult ure","Me OH","s or min","Cult ure","Me OH"]
["1.7 Samples taken and put into",null,null,null,null,1,null,30,20,null,30,20,null,30,20,null,30,20]
["    methanol on dry ice",null,null,null,null,2,null,30,20,null,30,20,null,30,20,null,30,20]
["1.11 Span",3,"g x1000",null,null,3,null,30,20,null,30,20,null,30,20,null,30,20]
["for",2,"min",null,null,4,null,30,20,null,30,20,null,30,20,null,30,20]
["1.11 Supernate removed",null,null,null,null,5,null,30,20,null,30,20,null,30,20,null,30,20]
["1.11 Resuspend in H2O",null,null,1,"mL",6,null,30,20,null,30,20,null,30,20,null,30,20]
["1.12 Transfered to screwcap tube",null,null,null,null,7,null,30,20,null,30,20,null,30,20,null,30,20]
["with",200,"µL zirconia",null,null,8,null,30,20,null,30,20,null,30,20,null,30,20]
["1.12 Span ",10,"sec  >13,000 x g",null,null,9,null,30,20,null,30,20,null,30,20,null,30,20]
["Supernate removed",null,null,null,null,10,null,30,20,null,30,20,null,30,20,null,30,20]
["Stored","Storage at -80 °C",null,null,null,11,null,30,20,null,30,20,null,30,20,null,30,20]
[null,null,null,null,null,12,null,30,20,null,30,20,null,30,20,null,30,20]
[null,null,null,null,null,13,null,30,20,null,30,20,null,30,20,null,30,20]
[null,null,null,null,null,14,null,30,20,null,30,20,null,30,20,null,30,20]

{"sheet":"Sect 2 RNA Prep","index":1,"n_rows":28,"n_cols":10,"rows_kept":28,"cut":false,"hidden":false,"merged":[[0,2,0,3],[0,4,0,5],[0,6,0,7],[0,8,0,9],[1,2,1,3],[1,4,1,5],[1,6,1,7],[1,8,1,9],[22,0,23,0],[2,2,2,3],[2,4,2,5],[2,6,2,7],[2,8,2,9]]}
["2. RNA Extraction",null,"Culture 1",null,"Culture 2",null,"Culture 3",null,"Culture 4"]
[null,"Date",{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"}]
[null,"Name"]
["2.2 S. pombe spike (Optional)","Vol",10,"µL",10,"µL",10,"µL",10,"µL"]
["2.4 AE buffer","Vol",400,"µL",400,"µL",400,"µL",400,"µL"]
["2.4 SDS [10%]","Vol",40,"µL",40,"µL",40,"µL",40,"µL"]
["2.6 Phenol, low pH","Vol",800,"µL",800,"µL",800,"µL",800,"µL"]
["2.7 Lyse in Beadbeater","For","3x","times","3x","times","3x","times","3x","times"]
["    rest in ice between","min",2,"min",2,"min",2,"min",2,"min"]
["2.8 Dry Ice","For",5,"min",5,"min",5,"min",5,"min"]
["2.8 Spin >13,000 x g (room temp)","For",5,"min",5,"min",5,"min",5,"min"]
[" Extract supernate to new tube"]
["2.9 Phenol:CHCl3 5:1, low pH","Vol",600,"µL",600,"µL",600,"µL",600,"µL"]
["vortex vigourously"]
["Spin >13,000 x g","For",5,"min",5,"min",5,"min",5,"min"]
["Extract supernate to new tube"]
["2.9 CHCl3","Vol",600,"µL",600,"µL",600,"µL",600,"µL"]
["vortex vigourously"]
["Spin >13,000 x g","For",5,"min",5,"min",5,"min",5,"min"]
["Extract supernate to new tube"]
["2.10 LiCl [10M]","Vol",300,"µL",300,"µL",300,"µL",300,"µL"]
["Mix"]
["Precipitate","Temp",4,"°C",4,"°C",4,"°C",4,"°C"]
[null,"For",10,"min",10,"min",10,"min",10,"min"]
["2.11 Spin >13,000 x g","For",5,"min",5,"min",5,"min",5,"min"]
["Wash 70% EtOH"]
["2.12 Resuspend 1x TE pH7.0","Vol",90,"µL",90,"µL",90,"µL",90,"µL"]
["Ensure complete resuspension and transfer to 0.2ml tube strip"]

{"sheet":"Sect 3 Biotinylation ","index":2,"n_rows":19,"n_cols":10,"rows_kept":19,"cut":false,"hidden":false,"merged":[[15,0,16,0],[9,0,11,0],[10,1,11,1],[0,2,0,3],[0,4,0,5],[1,2,1,3],[1,4,1,5],[2,2,2,3],[2,4,2,5],[2,6,2,7],[2,8,2,9],[1,8,1,9],[0,8,0,9],[0,6,0,7],[1,6,1,7]]}
["3. Biotinylation",null,"Culture 1",null,"Culture 2",null,"Culture 3",null,"Culture 4"]
[null,"Date",{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"}]
[null,"Name"]
["3.1 Begin incubation","Temp",65,"°C",65,"°C",65,"°C",65,"°C"]
["3.1 HPDP or MTS biotin","Vol",10,"µL",10,"µL",10,"µL",10,"µL"]
["Vortex "]
["Incubation","Temp",65,"°C",65,"°C",65,"°C",65,"°C"]
["  In dark eg PCR block","For",15,"min",15,"min",15,"min",15,"min"]
["3.2 Prepare Zeba columns during incubation"]
["Wash - spin 1500 x g, 1min remove fluid then add 1x TE pH7.0. Repeat 3 times","Vol",300,"µL",300,"µL",300,"µL",300,"µL"]
[null,"Washes","inital","wash 1","inital","wash 1","inital","wash 1","inital","wash 1"]
[null,null,"wash 2","wash 3","wash 2","wash 3","wash 2","wash 3","wash 2","wash 3"]
["3.3 After incubation put column in a new tube, add RNA"]
["Spin  1500 x g 2 min"]
["3.4 Precipitate","10M LiCl",40,"µL",40,"µL",40,"µL",40,"µL"]
["Precipitate","Temp",4,"°C",4,"°C",4,"°C",4,"°C"]
[null,"For",10,"min",10,"min",10,"min",10,"min"]
["Spin >13,000 x g","For",5,"min",5,"min",5,"min",5,"min"]
["3.5 & 3.6 Wash 80% EtOH",null,"wash 1","wash 2","wash 1","wash 2","wash 1","wash 2","wash 1","wash 2"]

{"sheet":"Sect 4 Beads","index":3,"n_rows":42,"n_cols":31,"rows_kept":42,"cut":false,"hidden":false,"merged":[[20,7,20,8],[21,7,21,8],[22,7,22,8],[23,7,23,8],[20,9,20,10],[21,9,21,10],[22,9,22,10],[23,9,23,10],[12,0,15,0],[10,1,10,2],[2,3,2,4],[2,5,2,6],[38,0,39,1],[25,0,26,1],[30,0,34,0],[35,0,36,1],[19,0,23,0],[19,1,23,1],[23,3,23,4],[20,5,20,6],[21,5,21,6],[22,5,22,6],[23,5,23,6],[0,9,0,10],[1,3,1,4],[1,5,1,6],[1,7,1,8],[1,9,1,10],[0,3,0,4],[0,5,0,6],[0,7,0,8],[1,13,2,13],[1,14,2,14],[1,15,2,15],[1,16,1,17],[37,3,37,4],[37,5,37,6],[37,7,37,8],[37,9,37,10],[2,9,2,10],[20,3,20,4],[21,3,21,4],[22,3,22,4],[4,0,4,10],[2,7,2,8],[5,0,7,0],[20,2,23,2],[1,21,1,22],[3,21,3,22],[4,21,5,22],[4,23,5,24],[0,17,0,18],[4,25,5,26],[4,27,5,28],[6,30,9,30],[3,16,3,19],[3,13,3,15]]}
["Beads",null,null,"Culture 1",null,"Culture 2",null,"Culture 3",null,"Culture 4",null,null,null,"4.2 Sample Volumes",null,null,"Date",{"$f":"TODAY()","v":"2019-05-14"},null,null,null,"4.5 Ammount of Bead Buffer to make up"]
[null,null,"Date",{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"},null,{"$f":"TODAY()","v":"2019-05-14"},null,null,null,"Typical sample vol",198,"µL","RNA to label",null,{"$f":"_xlfn.FLOOR.MATH((MIN(O6:O29)*(S3-(S3*0.21))/1000),0.01)","v":0},"mg",null,"#Samples",null,{"$f":"COUNT(O6:O29)","v":0}]
[null,null,"Name",null,null,null,null,null,null,null,null,null,null,null,null,null,"Total vol",null,250,"µL",null,"Minumum",null,{"$f":"X2*1.4","v":0},"mL","Minimum volume needed"]
["4.1 Redisolve the RNA in H2O",null,null,200,"µL",200,"µL",200,"µL",200,"µL",null,null,"Sample",null,null,"Add to sample (µL) (in this order)",null,null,null,null,"So make up",null,20,"mL","← Change this to have excess"]
["4.2 Measure the concentration of the RNA and adjust volumes to use. See form to right",null,null,null,null,null,null,null,null,null,null,null,null,"Sample name","conc µg/µL","vol to use (µL)","NaTM","NaPi","SDS","H2O",null,"Bead Buffer",null,"Stock Conc",null,"Final Conc",null,"Volume"]
["4.4 Add Beadbuffer","1st NaTM (10 x)","Vol",25,"µL",25,"µL",25,"µL",25,"µL",null,null,null,null,{"$f":"IF(O6=0,0,$S$2*1000/O6)","v":0},{"$f":"ROUND($S$3/10,1)","v":25},{"$f":"ROUND($S$3/10,1)","v":25},{"$f":"ROUND($S$3/100,1)","v":2.5},{"$f":"$S$3-SUM(P6:S6)","v":197.5}]
[null,"2nd 1 M NaPi","Vol",25,"µL",25,"µL",25,"µL",25,"µL",null,null,null,null,{"$f":"IF(O7=0,0,$S$2*1000/O7)","v":0},{"$f":"ROUND($S$3/10,1)","v":25},25,{"$f":"ROUND($S$3/100,1)","v":2.5},{"$f":"$S$3-SUM(P7:S7)","v":197.5},null,"H2O for 1x",null,null,"to",{"$f":"X4","v":20},"mL",{"$f":"X4-SUM(AB8:AB9)","v":16},"mL","1st","Add in this order"]
[null,"3rd 10% SDS","Vol",2.5,"µL",2.5,"µL",2.5,"µL",2.5,"µL",null,null,null,null,0,25,25,2.5,197.5,null,"NaTM, 10 x",null,10,"x",1,"x",{"$f":"$X$4*Z8/X8","v":2},"mL","2nd"]
["4.5 Make Bead Buffer, see form to far right",null,null,null,null,null,null,null,null,null,null,null,null,null,null,0,25,25,2.5,197.5,null,"NaPi pH 6.8",null,1,"M",0.1,"mM",{"$f":"$X$4*Z9/X9","v":2},"mL","3rd"]
["4.6 Bead aliquot",null,"Vol",50,"µL",50,"µL",50,"µL",50,"µL",null,null,null,null,0,25,25,2.5,197.5,null,"SDS",null,10,"%",0.1,"%",{"$f":"$X$4*Z10/X10","v":0.2},"mL","4th"]
["Type","NEB streptavidin",null,null,null,null,null,null,null,null,null,null,null,null,null,0,25,25,2.5,197.5]
["4.7 Wash ","Bead Buffer","Vol",200,"µL",200,"µL",200,"µL",200,"µL",null,null,null,null,0,25,25,2.5,197.5]
["4.8 Block","Bead Buffer","Vol",200,"µL",200,"µL",200,"µL",200,"µL",null,null,null,null,0,25,25,2.5,197.5]
[null,"Glycogen 20 mg/mL","Vol",10,"µL",10,"µL",10,"µL",10,"µL",null,null,null,null,0,25,25,2.5,197.5]
[null,"tRNA 5 mg/mL","Vol",2.5,"µL",2.5,"µL",2.5,"µL",2.5,"µL",null,null,null,null,0,25,25,2.5,197.5]
[null,"Time","For",20,"min",20,"min",20,"min",20,"min",null,null,null,null,0,25,25,2.5,197.5]
["Wash as 4.7 ","Bead Buffer","Vol",200,"µL",200,"µL",200,"µL",200,"µL",null,null,null,null,0,25,25,2.5,197.5,null,null,null,null,null,null,null," "]
["4.9 Add Sample",null,"Vol",250,"µL",250,"µL",250,"µL",250,"µL",null,null,null,null,0,25,25,2.5,197.5]
["Incubate",null,"For",30,"min",30,"min",30,"min",30,"min",null,null,null,null,0,25,25,2.5,197.5]
["4.10 and Wash as 4.7 with  Bead Buffer","1. On magnet\n2. pour off & aspirate \n3. add wash\n4. vortex\n5. Spin 1krpm a few sec","Vol",200,"µL",200,"µL",200,"µL",200,"µL",null,null,null,null,0,25,25,2.5,197.5]
[null,null,"repeat at least 3 times","wash 1",null,"wash 1",null,"wash 1",null,"wash 1",null,null,null,null,null,0,25,25,2.5,197.5]
[null,null,null,"wash 2",null,"wash 2",null,"wash 2",null,"wash 2",null,null,null,null,null,0,25,25,2.5,197.5]
[null,null,null,"wash 3",null,"wash 3",null,"wash 3",null,"wash 3",null,null,null,null,null,0,25,25,2.5,197.5]
[null,null,null,"wash 4",null,"wash 4",null,"wash 4",null,"wash 4",null,null,null,null,null,0,25,25,2.5,197.5]
["Other wash [Opt]",null,"Vol",0,"µL",0,"µL",0,"µL",0,"µL",null,null,null,null,0,25,25,2.5,197.5]
[null,null,null,"wash 1","wash 2","wash 1","wash 2","wash 1","wash 2","wash 1","wash 2",null,null,null,null,0,25,25,2.5,197.5]
[null,null,null,"wash 3","wash 4","wash 3","wash 4","wash 3","wash 4","wash 3","wash 4",null,null,null,null,0,25,25,2.5,197.5]
["4.13 Elution 1","BME 0.7 M","Vol",50,"µL",50,"µL",50,"µL",50,"µL",null,null,null,null,0,25,25,2.5,197.5]
["4.14 Elution 2","BME 0.7 M","Vol",50,"µL",50,"µL",50,"µL",50,"µL",null,null,null,null,0,25,25,2.5,197.5]
["4.15 Final bead removal, transfer to a freash tube"]
["4.16 Ethanol precipitate","4.10 Na acetate 3 M pH 5.3","Vol",10,"µL",10,"µL",10,"µL",10,"µL"]
[null,"4.10 Glycogen 20 mg/mL","Vol",1,"µL",1,"µL",1,"µL",1,"µL"]
[null,"Mix"]
[null,"Ethanol","Vol",280,"µL",280,"µL",280,"µL",280,"µL"]
[null,"Mix"]
["Precipitate",null,"Temp",-20,"°C",-20,"°C",-20,"°C",-20,"°C"]
[null,null,"For","O/N",null,"O/N",null,"O/N",null,"O/N"]
[null,null,"Date",{"$f":"TODAY()+1","v":"2019-05-15"},null,{"$f":"TODAY()+1","v":"2019-05-15"},null,{"$f":"TODAY()+1","v":"2019-05-15"},null,{"$f":"TODAY()+1","v":"2019-05-15"}]
["Spin     >13     krpm",null,"For",20,"min",20,"min",20,"min",20,"min"]
[null,null,"Temp",4,"°C",4,"°C",4,"°C",4,"°C"]
["4.17 Wash 70% EtOH"]
["4.18 Resuspend TE + RNase inhibitor",null,"Vol",10,"µL",10,"µL",10,"µL",10,"µL"]