Difference between revisions of "Team:IISER-Pune-India/InterLab"

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<h4>Fluorescence measurement were taken using Thermo Scientific Varioskan Flash plate reader.</h4>
 
<h4>Fluorescence measurement were taken using Thermo Scientific Varioskan Flash plate reader.</h4>
 
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<h1>Fluorescence measurement were taken using Thermo Scientific Varioskan Flash plate reader.</h1>
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<h2>Fluorescence measurement were taken using Thermo Scientific Varioskan Flash plate reader.</h2>
 
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Revision as of 22:09, 1 November 2017

Interlab

InterLab

Calibration Experiments

Materials:

  • 1ml LUDOX (provided in kit)
  • H20 (provided by team)
  • 96 well plate, black with flat, transparent/clear bottom preferred (provided by team)

Method

  • Add 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
  • Add 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
  • Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
  • Record the data in the table below or in your notebook
  • Import data into Excel (OD600 reference point tab)​ Sheet_1 provided

Materials:

  • fluorescein (provided in kit)
  • 10ml 1xPBS (phosphate buffered saline; provided by team)
  • Corning 96 well plate, black,flat bottom,half-well

Method:

Prepare the fluorescein stock solution:

  • Spin down fluorescein stock tube to make sure pellet is at the bottom of tube
  • Prepare 2x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS. [Note​: it is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip – if any particulates are visible in the pipette tip continue to mix the solution until they disappear.
  • Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 µM (500µL of 2x fluorescein in 500 µL 1x PBS will make 1 mL of 50 µM (1x) fluorescein solution.)

Prepare the serial dilutions of fluorescein:

  • Add 100 µl of PBS​ into wells A2, B2, C2, D2....A12, B12, C12, D12
  • Add 200 µl​ of fluorescein 1x stock​ solution into A1, B1, C1, D1
  • Transfer 100 µl of fluorescein stock solution from A1 into A2.
  • Mix A2 by pipetting up and down 3x and transfer 100 µl into A3…
  • Mix A3 by pipetting up and down 3x and transfer 100 µl into A4...
  • Mix A4 by pipetting up and down 3x and transfer 100 µl into A5...
  • Mix A5 by pipetting up and down 3x and transfer 100 µl into A6...
  • Mix A6 by pipetting up and down 3x and transfer 100 µl into A7...
  • Mix A7 by pipetting up and down 3x and transfer 100 µl into A8...
  • Mix A8 by pipetting up and down 3x and transfer 100 µl into A9...
  • Mix A9 by pipetting up and down 3x and transfer 100 µl into A10...
  • Mix A10 by pipetting up and down 3x and transfer 100 µl into A11...
  • Mix A11 by pipetting up and down 3x and transfer 100 µl into liquid waste
  • Repeat dilution series for rows B, C
  • Measure fluorescence of all samples in all standard measurement modes in instrumen
  • Record the data in your noteboo
  • Import data into Excel (fluorescein standard curve tab)​ Sheet_1 provided

Fluorescence measurement were taken using Thermo Scientific Varioskan Flash plate reader.

Fluorescence measurement were taken using Thermo Scientific Varioskan Flash plate reader.

  • Plate reader settings:
  • Light source: Xenon flash lamp
  • Excitation wavelength-480nm
  • Emission wavelength-510nm
  • Measurement time-100ms
  • Excitation bandwidth-5nm
  • Emission bandwidth-12nm
LB liquid medium: 1000mL
Experimental Values: Raw Abs600         Raw Fluorescence                                              
Sample set: Replicate 1 Replicate 2 Replicate 3 Replicate 4   Replicate 1 Replicate 2 Replicate 3 Replicate 4                                        
Blank media 0.039 0.038 0.038 0.038   11.92 11.32 11.99 11.91                                        
Blank mean: 0.03825         11.785         OD - Background         Fluorescence - Background         uM Fluorescein / OD600         Summary Statistics      
Hour 0:                     Replicate 1 Replicate 2 Replicate 3 Replicate 4   Replicate 1 Replicate 2 Replicate 3 Replicate 4   Replicate 1 Replicate 2 Replicate 3 Replicate 4   Arith. Mean Arith. Std.Dev. Geo. Mean Geo. Std. Dev.
Negative Control (Colony 1) 0.047 0.047 0.047 0.047   12.75 12.83 13.35 12.82   0.00875 0.00875 0.00875 0.00875   0.965 1.045 1.565 1.035   0.10 0.10 0.16 0.10   0.12 0.03 0.11 1.246
Negative Control (Colony 2) 0.047 0.047 0.047 0.046   10.89 9.781 7.717 12.44   0.00875 0.00875 0.00875 0.00775   -0.895 -2.004 -4.068 0.655   -0.09 -0.20 -0.41 0.07   -0.16 0.20 #NUM! #NUM!
Positive Control (Colony 1) 0.047 0.048 0.047 0.047   12.13 12.44 13.99 13.51   0.00875 0.00975 0.00875 0.00875   0.345 0.655 2.205 1.725   0.03 0.06 0.22 0.17   0.12 0.09 0.09 2.410
Positive Control (Colony 2) 0.049 0.047 0.046 0.046   13.46 12.61 13.06 13.77   0.01075 0.00875 0.00775 0.00775   1.675 0.825 1.275 1.985   0.14 0.08 0.14 0.22   0.15 0.06 0.14 1.505
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) 0.049 0.05 0.048 0.049   18.19 20.69 19.15 19.37   0.01075 0.01175 0.00975 0.01075   6.405 8.905 7.365 7.585   0.52 0.66 0.66 0.62   0.62 0.07 0.61 1.119
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) 0.047 0.046 0.047 0.046   21.62 19.76 20.76 18.28   0.00875 0.00775 0.00875 0.00775   9.835 7.975 8.975 6.495   0.99 0.90 0.90 0.73   0.88 0.10 0.88 1.132
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) 0.047 0.046 0.047 0.047   13.95 14.37 14.62 14.54   0.00875 0.00775 0.00875 0.00875   2.165 2.585 2.835 2.755   0.22 0.29 0.28 0.28   0.27 0.03 0.27 1.147
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) 0.048 0.047 0.046 0.047   14.38 16.27 13.38 15.53   0.00975 0.00875 0.00775 0.00875   2.595 4.485 1.595 3.745   0.23 0.45 0.18 0.38   0.31 0.12 0.29 1.523
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) 0.048 0.048 0.071 0.049   11.44 12.69 14.09 11.64   0.00975 0.00975 0.03275 0.01075   -0.345 0.905 2.305 -0.145   -0.03 0.08 0.06 -0.01   0.03 0.05 #NUM! #NUM!
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) 0.047 0.046 0.045 0.051   13.57 13.91 12.65 13.24   0.00875 0.00775 0.00675 0.01275   1.785 2.125 0.865 1.455   0.18 0.24 0.11 0.10   0.16 0.06 0.15 1.504
Test Device 4: J23101+I13504 (Colony 1) 0.045 0.046 0.047 0.047   14.09 13.1 14.81 13.72   0.00675 0.00775 0.00875 0.00875   2.305 1.315 3.025 1.935   0.30 0.15 0.30 0.19   0.24 0.08 0.23 1.416
Test Device 4: J23101+I13504 (Colony 2) 0.048 0.046 0.046 0.046   15.34 14.29 13.91 13.42   0.00975 0.00775 0.00775 0.00775   3.555 2.505 2.125 1.635   0.32 0.28 0.24 0.18   0.26 0.06 0.25 1.267
Test Device 5: J23106+I13504 (Colony 1) 0.046 0.046 0.047 0.046   13.8 11.54 12.31 10.5   0.00775 0.00775 0.00875 0.00775   2.015 -0.245 0.525 -1.285   0.23 -0.03 0.05 -0.15   0.03 0.16 #NUM! #NUM!
Test Device 5: J23106+I13504 (Colony 2) 0.047 0.047 0.046 0.046   13.06 14.03 13.71 13.77   0.00875 0.00875 0.00775 0.00775   1.275 2.245 1.925 1.985   0.13 0.22 0.22 0.22   0.20 0.05 0.19 1.320
Test Device 6: J23117+I13504 (Colony 1) 0.046 0.047 0.047 0.047   13.49 12.07 12.23 12.83   0.00775 0.00875 0.00875 0.00875   1.705 0.285 0.445 1.045   0.19 0.03 0.04 0.10   0.09 0.07 0.07 2.353
Test Device 6: J23117+I13504 (Colony 2) 0.046 0.044 0.044 0.044   13.24 12.34 13.56 13.93   0.00775 0.00575 0.00575 0.00575   1.455 0.555 1.775 2.145   0.16 0.08 0.27 0.33   0.21 0.11 0.19 1.829
                                                           
Hour 2:                                                          
Negative Control (Colony 1) 0.055 0.068 0.054 0.053   13.36 12.83 12.3 12.85   0.01675 0.02975 0.01575 0.01475   1.575 1.045 0.515 1.065   0.08 0.03 0.03 0.06   0.05 0.03 0.05 1.690
Negative Control (Colony 2) 0.232 0.209 0.148 0.054   13.54 12.85 13.88 11.16   0.19375 0.17075 0.10975 0.01575   1.755 1.065 2.095 -0.625   0.01 0.01 0.02 -0.03   0.00 0.02 #NUM! #NUM!
Positive Control (Colony 1) 0.058 0.053 0.053 0.052   20.39 19.68 20.97 21   0.01975 0.01475 0.01475 0.01375   8.605 7.895 9.185 9.215   0.38 0.47 0.55 0.59   0.50 0.09 0.49 1.210
Positive Control (Colony 2) 0.152 0.104 0.053 0.054   21.64 20.63 19.6 20.02   0.11375 0.06575 0.01475 0.01575   9.855 8.845 7.815 8.235   0.08 0.12 0.46 0.46   0.28 0.21 0.21 2.540
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) 0.051 0.054 0.049 0.049   26.75 29.96 29.37 29.77   0.01275 0.01575 0.01075 0.01075   14.965 18.175 17.585 17.985   1.03 1.01 1.43 1.47   1.24 0.25 1.22 1.226
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) 0.204 0.193 0.195 0.136   29.84 32.35 33.21 26.81   0.16575 0.15475 0.15675 0.09775   18.055 20.565 21.425 15.025   0.10 0.12 0.12 0.13   0.12 0.02 0.12 1.154
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) 0.052 0.05 0.05 0.05   31.21 29.42 29.86 31.01   0.01375 0.01175 0.01175 0.01175   19.425 17.635 18.075 19.225   1.24 1.32 1.35 1.43   1.33 0.08 1.33 1.063
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) 0.054 0.076 0.053 0.052   30.14 35.8 34.28 31.55   0.01575 0.03775 0.01475 0.01375   18.355 24.015 22.495 19.765   1.02 0.56 1.34 1.26   1.04 0.35 0.99 1.491
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) 0.054 0.055 0.057 0.054   14.69 14.47 16.36 16.08   0.01575 0.01675 0.01875 0.01575   2.905 2.685 4.575 4.295   0.16 0.14 0.21 0.24   0.19 0.05 0.18 1.278
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) 0.055 0.054 0.053 0.052   17.35 16.78 18.05 15.31   0.01675 0.01575 0.01475 0.01375   5.565 4.995 6.265 3.525   0.29 0.28 0.37 0.22   0.29 0.06 0.29 1.230
Test Device 4: J23101+I13504 (Colony 1) 0.054 0.053 0.052 0.052   21.9 22.44 23.34 23.84   0.01575 0.01475 0.01375 0.01375   10.115 10.655 11.555 12.055   0.56 0.63 0.74 0.77   0.68 0.09 0.67 1.154
Test Device 4: J23101+I13504 (Colony 2) 0.051 0.052 0.051 0.05   25.2 25.93 25.8 24.39   0.01275 0.01375 0.01275 0.01175   13.415 14.145 14.015 12.605   0.92 0.90 0.96 0.94   0.93 0.03 0.93 1.029
Test Device 5: J23106+I13504 (Colony 1) 0.055 0.053 0.051 0.053   22.94 22.74 21.48 21.35   0.01675 0.01475 0.01275 0.01475   11.155 10.955 9.695 9.565   0.58 0.65 0.67 0.57   0.62 0.05 0.62 1.082
Test Device 5: J23106+I13504 (Colony 2) 0.057 0.056 0.055 0.055   22.02 22.25 22.51 21.09   0.01875 0.01775 0.01675 0.01675   10.235 10.465 10.725 9.305   0.48 0.52 0.56 0.49   0.51 0.04 0.51 1.075
Test Device 6: J23117+I13504 (Colony 1) 0.053 0.054 0.056 0.055   16.19 15.64 16.24 16.07   0.01475 0.01575 0.01775 0.01675   4.405 3.855 4.455 4.285   0.26 0.21 0.22 0.22   0.23 0.02 0.23 1.094
Test Device 6: J23117+I13504 (Colony 2) 0.051 0.051 0.049 0.05   16.13 16.2 16.26 17.05   0.01275 0.01275 0.01075 0.01175   4.345 4.415 4.475 5.265   0.30 0.30 0.36 0.39   0.34 0.05 0.34 1.145
                                                           
Hour 4:                                                          
Negative Control (Colony 1) 0.191 0.15 0.134 0.139   14.45 15.48 14.63 13.73   0.15275 0.11175 0.09575 0.10075   2.665 3.695 2.845 1.945   0.02 0.03 0.03 0.02   0.02 0.01 0.02 1.370
Negative Control (Colony 2) 0.147 0.161 0.151 0.142   14.89 15.77 16.32 15.74   0.10875 0.12275 0.11275 0.10375   3.105 3.985 4.535 3.955   0.03 0.03 0.04 0.03   0.03 0.00 0.03 1.169
Positive Control (Colony 1) 0.184 0.139 0.13 0.127   50.64 49.12 44.43 39.3   0.14575 0.10075 0.09175 0.08875   38.855 37.335 32.645 27.515   0.23 0.32 0.31 0.27   0.29 0.04 0.28 1.161
Positive Control (Colony 2) 0.157 0.14 0.133 0.134   58.28 57.94 52.46 52.37   0.11875 0.10175 0.09475 0.09575   46.495 46.155 40.675 40.585   0.34 0.40 0.38 0.37   0.37 0.02 0.37 1.063
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) 0.057 0.056 0.055 0.054   48.17 41.93 44.44 43.61   0.01875 0.01775 0.01675 0.01575   36.385 30.145 32.655 31.825   1.70 1.49 1.71 1.77   1.67 0.12 1.66 1.079
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) 0.056 0.054 0.054 0.052   46.69 50.03 47.27 45.78   0.01775 0.01575 0.01575 0.01375   34.905 38.245 35.485 33.995   1.72 2.13 1.97 2.17   2.00 0.20 1.99 1.110
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) 0.143 0.13 0.129 0.116   76.83 75.98 72.69 70.29   0.10475 0.09175 0.09075 0.07775   65.045 64.195 60.905 58.505   0.54 0.61 0.59 0.66   0.60 0.05 0.60 1.084
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) 0.131 0.106 0.1 0.096   75.71 70.61 69.71 75.01   0.09275 0.06775 0.06175 0.05775   63.925 58.825 57.925 63.225   0.60 0.76 0.82 0.96   0.79 0.15 0.78 1.213
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) 0.208 0.152 0.144 0.126   18.56 17.22 16.66 15.38   0.16975 0.11375 0.10575 0.08775   6.775 5.435 4.875 3.595   0.03 0.04 0.04 0.04   0.04 0.00 0.04 1.092
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) 0.155 0.131 0.114 0.115   16.97 16.11 16.05 17   0.11675 0.09275 0.07575 0.07675   5.185 4.325 4.265 5.215   0.04 0.04 0.05 0.06   0.05 0.01 0.05 1.214
Test Device 4: J23101+I13504 (Colony 1) 0.115 0.118 0.099 0.1   46.38 44.29 43.31 42.04   0.07675 0.07975 0.06075 0.06175   34.595 32.505 31.525 30.255   0.40 0.36 0.45 0.43   0.41 0.04 0.41 1.111
Test Device 4: J23101+I13504 (Colony 2) 0.107 0.103 0.099 0.094   46.3 44.96 44.81 42.31   0.06875 0.06475 0.06075 0.05575   34.515 33.175 33.025 30.525   0.44 0.45 0.48 0.48   0.46 0.02 0.46 1.044
Test Device 5: J23106+I13504 (Colony 1) 0.119 0.112 0.122 0.117   24.84 25.74 27.41 27.58   0.08075 0.07375 0.08375 0.07875   13.055 13.955 15.625 15.795   0.14 0.17 0.16 0.18   0.16 0.01 0.16 1.096
Test Device 5: J23106+I13504 (Colony 2) 0.139 0.125 0.147 0.135   24.09 22.96 26.29 26.06   0.10075 0.08675 0.10875 0.09675   12.305 11.175 14.505 14.275   0.11 0.11 0.12 0.13   0.12 0.01 0.12 1.083
Test Device 6: J23117+I13504 (Colony 1) 0.137 0.145 0.142 0.125   13.65 14.24 13.76 14.66   0.09875 0.10675 0.10375 0.08675   1.865 2.455 1.975 2.875   0.02 0.02 0.02 0.03   0.02 0.01 0.02 1.301
Test Device 6: J23117+I13504 (Colony 2) 0.11 0.111 0.11 0.116   14.42 14.08 13.93 14.6   0.07175 0.07275 0.07175 0.07775   2.635 2.295 2.145 2.815   0.03 0.03 0.03 0.03   0.03 0.00 0.03 1.107
                                                           
Hour 6:                                                          
Negative Control (Colony 1) 0.403 0.396 0.385 0.384   13.86 14.13 14.1 13.53   0.36475 0.35775 0.34675 0.34575   2.075 2.345 2.315 1.745   0.00 0.01 0.01 0.00   0.01 0.00 0.01 1.140
Negative Control (Colony 2) 0.282 0.399 0.446 0.312   13.67 13.87 14.02 13.94   0.24375 0.36075 0.40775 0.27375   1.885 2.085 2.235 2.155   0.01 0.01 0.00 0.01   0.01 0.00 0.01 1.209
Positive Control (Colony 1) 0.308 0.279 0.297 0.282   84.88 74.53 79.93 79.25   0.26975 0.24075 0.25875 0.24375   73.095 62.745 68.145 67.465   0.24 0.23 0.23 0.24   0.23 0.01 0.23 1.028
Positive Control (Colony 2) 0.393 0.303 0.237 0.411   79.7 68.25 81.04 81.93   0.35475 0.26475 0.19875 0.37275   67.915 56.465 69.255 70.145   0.17 0.19 0.31 0.16   0.21 0.07 0.20 1.336
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) 0.069 0.059 0.068 0.057   53.36 48.94 53.32 52.33   0.03075 0.02075 0.02975 0.01875   41.575 37.155 41.535 40.545   1.19 1.57 1.22 1.90   1.47 0.33 1.44 1.248
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) 0.07 0.069 0.068 0.068   54.14 55.06 56.59 55.34   0.03175 0.03075 0.02975 0.02975   42.355 43.275 44.805 43.555   1.17 1.23 1.32 1.28   1.25 0.07 1.25 1.054
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) 0.308 0.307 0.288 0.324   146.1 129 142.7 146.6   0.26975 0.26875 0.24975 0.28575   134.315 117.215 130.915 134.815   0.44 0.38 0.46 0.41   0.42 0.03 0.42 1.082
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) 0.403 0.427 0.217 0.287   125.9 120.6 132.6 121.6   0.36475 0.38875 0.17875 0.24875   114.115 108.815 120.815 109.815   0.27 0.25 0.59 0.39   0.37 0.16 0.35 1.487
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) 0.398 0.399 0.392 0.343   16.24 15.33 14.79 16.13   0.35975 0.36075 0.35375 0.30475   4.455 3.545 3.005 4.345   0.01 0.01 0.01 0.01   0.01 0.00 0.01 1.261
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) 0.3 0.295 0.209 0.181   15.95 15.66 15.93 15.44   0.26175 0.25675 0.17075 0.14275   4.165 3.875 4.145 3.655   0.01 0.01 0.02 0.02   0.02 0.00 0.02 1.318
Test Device 4: J23101+I13504 (Colony 1) 0.272 0.274 0.264 0.264   87.02 86.88 85.79 83.04   0.23375 0.23575 0.22575 0.22575   75.235 75.095 74.005 71.255   0.28 0.28 0.29 0.28   0.28 0.00 0.28 1.016
Test Device 4: J23101+I13504 (Colony 2) 0.296 0.236 0.225 0.228   97.47 97.03 97.78 82.43   0.25775 0.19775 0.18675 0.18975   85.685 85.245 85.995 70.645   0.29 0.38 0.40 0.33   0.35 0.05 0.35 1.158
Test Device 5: J23106+I13504 (Colony 1) 0.36 0.366 0.368 0.347   47.54 46.09 45.93 42.66   0.32175 0.32775 0.32975 0.30875   35.755 34.305 34.145 30.875   0.10 0.09 0.09 0.09   0.09 0.00 0.09 1.045
Test Device 5: J23106+I13504 (Colony 2) 0.327 0.324 0.294 0.309   36.82 41.73 41.66 37.26   0.28875 0.28575 0.25575 0.27075   25.035 29.945 29.875 25.475   0.08 0.09 0.10 0.08   0.09 0.01 0.09 1.138
Test Device 6: J23117+I13504 (Colony 1) 0.387 0.402 0.38 0.343   14.5 14.72 14.73 14.63   0.34875 0.36375 0.34175 0.30475   2.715 2.935 2.945 2.845   0.01 0.01 0.01 0.01   0.01 0.00 0.01 1.083
Test Device 6: J23117+I13504 (Colony 2) 0.351 0.374 0.356 0.375   14.53 14.82 14.32 14.82   0.31275 0.33575 0.31775 0.33675   2.745 3.035 2.535 3.035   0.01 0.01 0.01 0.01   0.01 0.00 0.01 1.060
LB agar plates: (~45-50 plates)

Requirements

  • 2L Erlenmeyer flask
  • LB broth powder
  • Bacteriological Agar
  • Distilled or milliQ water
  • Magnetic stirrer and bead(optional)
  • Measuring cylinder
  • Autoclave tape
  • Cotton plug(for the flask)
  • Aluminium foil
  • Autoclaving machine
  • Petri plates

Procedure

  1. Prepare 1L of LB liquid (25g broth powder per 1L) in a 2000mL Erlenmeyer flask.
  2. Add 15g (1.5%) bacteriological agar
  3. Use magnetic stirrer and bead to mix , if not available simply swirl to mix. Powder will not dissolve completely.
  4. Make a nice cotton plug by rolling and folding a big piece of cotton and put it on the mouth of the flask(Try pulling it out once, if it makes a noise then, Bravo!)
  5. Add a fresh piece of aluminum foil to cover the top, and add a fresh piece of autoclave tape.
  6. Autoclave at 121°C
  7. After cycle is complete, remove to benchtop to cool to ~50 C. (As a ballpark, this is still quite warm, but cool enough to leave your gloved hand on the outside of the flask for several seconds without burning it. The media should still be liquid but should be just beginning to thicken)
  8. If you are adding antibiotics, add them at this point, and swirl the flask vigorously for several seconds to mix (1000 times dilution of the stock solution)
  9. Set out your plates, and label the bottom with the appropriate antibiotic: A=ampicillin, C=chloramphenicol, K=kanamycin, T=tetracycline, S=streptomycin
  10. Pour out the media into petri dishes as follows: Remove the lid to a dish, and remove the foil cover to the flask. Pour just enough LB agar into the petri dish to completely cover the bottom of the dish, then replace the lid.
  11. Finish pouring dishes within 5 minutes of adding the antibiotic.
  12. Dishes should be stored UPSIDE DOWN in the cold room.
SOB medium: (250mL)

Requirements

  • 1L autoclave bottle or erlenmeyer flask
  • 6.25g LB broth powder
  • 2.5mM KCl
  • 20mM MgSO 4 .7H 2 O
  • Distilled or milliQ water
  • Magnetic stirrer and bead(optional)
  • Measuring cylinder
  • Autoclave tape
  • Cotton plug(for the flask)
  • Aluminium foil
  • Autoclaving machine

Procedure

  1. In a 1L autoclave bottle or flask , add 6.25g LB broth powder.
  2. Calculate and weigh 2.5mM KCl and 20mM MgSO 4 .7H 2 O.
  3. Adjust pH to 7.5(using NaOH)
  4. Add distilled water to make the solution upto 250mL.
  5. Either Swirl or use magnetic stirrer and a bead to mix(might not dissolve completely) .
  6. Make a nice cotton plug by rolling and folding a big piece of cotton and put it on the mouth of the flask.(Try pulling it out once, if it makes a noise then, Bravo!)
  7. Put an Aluminium foil on top of it and a piece of autoclave tape
  8. Keep the cap slightly open if using a bottle.
  9. Autoclave at 121°C
  10. Cool to RT before use. Do not tighten cap until cool.
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