Difference between revisions of "Team:KU Leuven/InterLabStudy"

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Competent cells
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                <p class="lead">Plate Top10 cells and incubate at 37ºC overnight</p>
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              </li>
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              <li>
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                <p class="lead">Pick one colony, inoculate in LB media and incubate overnight while shaking at 37ºC</p>
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              </li>
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              <li>
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                <p class="lead">Dilute the culture in fresh medium, and continue the incubation until the OD<sub>600</sub>= 0.4-0.6</p>
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              </li>
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              <li>
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                <p class="lead">Centrifugation 5 min. at 4000 rpm.</p>
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              </li>
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              <li>
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                <p class="lead">Pellet cells and resuspend in 100mM of CaCl<sub>2</sub> solution</p>
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              </li>
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              <li>
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                <p class="lead">Incubate on ice for 20 min</p>
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              </li>
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              <li>
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                <p class="lead">Centrifugation 5 min at 3000 rpm</p>
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              </li>
 +
 +
              <li>
 +
                <p class="lead">Pellet cells and suspend again in 100mM CaCl<sub>2</sub> solution</p>
 +
              </li>
 +
 +
              <li>
 +
                <p class="lead">Incubate on ice for 60 min</p>
 +
              </li>
 +
 +
              <li>
 +
                <p class="lead">Centrifugation 5 min at 3000 rpm</p>
 +
              </li>
 +
 +
              <li>
 +
                <p class="lead">Add a solution of 100mM CaCl<sub>2</sub> + 40% glycerol</p>
 +
              </li>
 +
 +
              <li>
 +
                <p class="lead">Store immediately at -80ºC</p>
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              </li>
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            </ol>
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          </div>
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Revision as of 15:45, 6 September 2015

Logo


Interlab study

Results

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Table 1 Fluorescence measurements


D1, D2, D3 Devices J23117 + I13504, J2310 + I13504 and J23101 + I13504 respectively


LB + Cam LB+Cam+Cells D1 D2 D3
512 601 737 685 751 9425 9322 9737 22786 25895 25048
546 594 754 641 641 9669 9545 9876 23159 26460 24785
549 587 722 660 697 9407 9321 9677 22719 25931 24935

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Table 2 Absorbance measurements


D1, D2, D3 Devices J23117 + I13504, J2310 + I13504 and J23101 + I13504 respectively


LB + Cam LB+Cam+Cells D1 D2 D3
0.0434 0.2052 0.2174 0.1942 0.2201 0.2088 0.2082 0.2154 0.2139 0.2210 0.2218
0.0405 0.2000 0.2182 0.1974 0.1980 0.2108 0.2111 0.2123 0.2143 0.2179 0.2056
0.0379 0.2064 0.2141 0.1961 0.2036 0.2137 0.2117 0.2164 0.2202 0.2261 0.2083

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nunc nibh ligula, faucibus a elit id, egestas posuere est. Donec cursus enim non lacus dignissim, id vulputate leo tincidunt. Aliquam pretium luctus augue, nec tempor odio sagittis eu. Morbi rutrum suscipit purus a iaculis. Suspendisse in augue eget turpis volutpat bibendum. Vivamus vel libero ac velit pulvinar auctor in eu nunc. Duis urna lectus, cursus ac tempor sed, lobortis id tellus.


Table 3 Values for Biological replicates


D1, D2, D3 Devices J23117 + I13504, J2310 + I13504 and J23101 + I13504 respectively

B1, B2, B3 Biological replicates of the respective devices

T1, T2, T3 Technical replicates


D1 D2 D3 LB+Cam+Cells
T1 T2 T3 T1 T2 T3 T1 T2 T2
B1 3390 3456 3372 45139 45868 44020 106526 108068 103174 2929
B2 3527 3247 3366 44774 45216 44029 117172 121432 114688 2970
B3 3399 3237 3426 45204 46519 44718 112931 120550 119707 2844
Brep Avg 3439 3313 3388 45039 45868 44256 112210 116683 112523
BRep SD 270 101 162 189 532 327 4376 6102 6921

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nunc nibh ligula, faucibus a elit id, egestas posuere est. Donec cursus enim non lacus dignissim, id vulputate leo tincidunt. Aliquam pretium luctus augue, nec tempor odio sagittis eu. Morbi rutrum suscipit purus a iaculis. Suspendisse in augue eget turpis volutpat bibendum. Vivamus vel libero ac velit pulvinar auctor in eu nunc. Duis urna lectus, cursus ac tempor sed, lobortis id tellus.


Table 4 Values for Technical replicates


D1, D2, D3 Devices J23117 + I13504, J2310 + I13504 and J23101 + I13504 respectively

B1, B2, B3 Biological replicates of the respective devices

T1, T2, T3 Technical replicates


D1 D2 D3 LB+Cam+ Cells
B1 B2 B3 B1 B2 B3 B1 B2 B3
T1 3390 3527 3399 45139 44774 45204 106526 117172 112931 2929
T2 3456 3247 3237 45868 45216 46519 108068 121432 120550 2970
T3 3372 3366 3426 44020 44029 44718 103174 114688 119707 2844
TRep Avg 3406 3380 3354 45009 44673 45480 105923 117764 117729 2914
TRep SD 36 115 140 760 490 761 2043 2785 3410 52

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nunc nibh ligula, faucibus a elit id, egestas posuere est. Donec cursus enim non lacus dignissim, id vulputate leo tincidunt. Aliquam pretium luctus augue, nec tempor odio sagittis eu. Morbi rutrum suscipit purus a iaculis. Suspendisse in augue eget turpis volutpat bibendum. Vivamus vel libero ac velit pulvinar auctor in eu nunc. Duis urna lectus, cursus ac tempor sed, lobortis id tellus.


Table 5 Standard graph


Fluorescein standard
concentration (ng/mL) F1 F2 F3 average F average F' SD
0 9 13 12 11 0 1.699673171
10 1140 1168 1151 1153 1142 11.5181017
25 2543 2600 2631 2591 2580 36.4447832
50 4822 5097 5154 5024 5013 144.9513328
125 12879 12902 12931 12904 12893 21.27596453
250 25494 25761 25765 25673 25662 126.8183303
375 37474 37505 37899 37626 37615 193.4545597
500 46698 49051 48798 48182 48171 1054.652023

Figure 1
Standard curve for fluorescein

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nunc nibh ligula, faucibus a elit id, egestas posuere est. Donec cursus enim non lacus dignissim, id vulputate leo tincidunt. Aliquam pretium luctus augue, nec tempor odio sagittis eu. Morbi rutrum suscipit purus a iaculis. Suspendisse in augue eget turpis volutpat bibendum. Vivamus vel libero ac velit pulvinar auctor in eu nunc. Duis urna lectus, cursus ac tempor sed, lobortis id tellus.


Table 6 Extrapolation of GFP concentration using fluorescence measurements and fluorescein standard


D1 D2 D3
Brep Avg 3439 3313 3388 45039 45868 44256 112210 116683 112523
(Brep Avg) - (LB+cam+cells avg) 525 399 474 42125 42954 41342 109296 113769 109609
Concentration (ng/mL) 1.929 0.639 1.407 427.88 436.36 419.88 1115.64 1161.44 1118.85
TRep Avg 3406 3380 3354 45009 44673 45480 105923 117764 117729
(Trep avg) - (LB+cam+cellsavg) 492 466 440 42095 41759 42566 103009 114850 114815
Concentration (ng/mL) 1.591 1.325 1.059 427.56 424.12 432.39 1051.27 1172.51 1172.15

Protocols





  1. Plate Top10 cells and incubate at 37ºC overnight

  2. Pick one colony, inoculate in LB media and incubate overnight while shaking at 37ºC

  3. Dilute the culture in fresh medium, and continue the incubation until the OD600= 0.4-0.6

  4. Centrifugation 5 min. at 4000 rpm.

  5. Pellet cells and resuspend in 100mM of CaCl2 solution

  6. Incubate on ice for 20 min

  7. Centrifugation 5 min at 3000 rpm

  8. Pellet cells and suspend again in 100mM CaCl2 solution

  9. Incubate on ice for 60 min

  10. Centrifugation 5 min at 3000 rpm

  11. Add a solution of 100mM CaCl2 + 40% glycerol

  12. Store immediately at -80ºC


Contact

Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone n°: +32(0)16 32 73 19