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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;By inserting the final system of our project, we can create a sensor which will produce a color gradient after approaching to the nitrites and nitrates. From this goal we are willing to reach, we can observe the visual change of the bacteria easily, and simultaneously receive a beautiful and cheerful ending of our whole project in the IGEM competition.<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;By inserting the final system of our project, we can create a sensor which will produce a color gradient after approaching to the nitrites and nitrates. From this goal we are willing to reach, we can observe the visual change of the bacteria easily, and simultaneously receive a beautiful and cheerful ending of our whole project in the IGEM competition.<br>
 +
SZMS-iGEM    Protocol<br>
 +
0. First of all, be prepared for the plates.(Resistance selection medium ,sterilization)<br>
 +
Part One  Kitting and Incubating( LEAST 2+12 hours)<br>
 +
1. Find parts we want from 384 panel in the refrigerator. The parts we found and decided to use were 1-18C (BBa_J23100) and 2-2I (BBa_J23111), which were not used.<br>
 +
2. Inside the plate there were DNA powder, we used tips of trace extractor to puncture aluminum foil, and draw 10μL double-distilled(DD) water with the trace extractor. dissolve DNA into solution. suck the solution again, put into the Polypropylene centrifuge tube (before this we should always write its tab on)<br>
 +
---------------------------------------------------15 minutes waiting---------------------------------------------------<br>
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3. Get the competent cell from the -73C fridge with the commending of our mentors.<br>
 +
4. Draw the DNA solution into the tube of E.coli (by trace extraction) 2μL out of 10μL, and put it inside the ice box for 30 minutes.<br>
 +
--------------------------------------------------30 minutes waiting----------------------------------------------------<br>
 +
5. Turn on the Thermomixer comfort 10min before using.<br>
 +
6. Put E.coli tube into Thermomixer comfort for 42 Celsius degree and 90 seconds “Hot Shock”.<br>
 +
-------------------------------------------------90 seconds waiting------------------------------------------------------<br>
 +
7. Put E.coli back into the ice-box for 5 minutes.<br>
 +
---------------------------------------------------5 minutes waiting-----------------------------------------------------<br>
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8. Add 0.5mL LB (Luria-Bertani medium) into E.coli tube.<br>
 +
9. Get 4 culture medium after the tubes were shaken in Thermostatic oscillation incubator.<br>
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10. Use spreader to put E.coli on the plate (inside super clean bench and remember to grill the spreader on the alcohol burner before using).<br>
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11. Take the plates with E.coli outside and start germiculturing them in the Constant temperature incubator (37 Celsius degree).<br>
 +
<br>
 +
Part Two  Selecting and Retaining(LEAST 1+12 hours)<br>
 +
12. Take the plates out of the Constant temperature incubator.<br>
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13. Take the test tube from the super clean bench.<br>
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14. Add 0.5mL LB and 200X of antibiotic into the tubes.<br>
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15. Select the strain used in the experiment from the plate (the one which is integral and independent with lucid edge will be the proper choice), and put it into the tubes with tips of trace extractor.<br>
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16. Clean the super clean incubator, put the plates back into the Constant temperature incubator.<br>
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17. Put the tubes into the shaker and shake under the temperature of 37 Celsius degree.<br>
 +
<br>
 +
Part Three  Measuring(LEAST 2 hours)<br>
 +
18. Drop one μL of buffer on the probe as a blank control group.<br>
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19. Clean the probe and drop one μL of DNA solution on it.<br>
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20. Start the procedure.<br>
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21. Note the concentration showed on the screen.
 +
※The concentration we noted was 260/280: 1.93, 44.2ng/μL
 +
The ideal data of concentration 260/280 is 1.8-2.0
 +
Protein pollution if lower than 1.8 and ion pollution when higher than 2.0<br>
 +
<br>
 +
Part Four  Plasmid Collecting(LEAST 3 hours)<br>
 +
22. Select the strain (As 15)<br>
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23. Retain the backups of the bacteria (Into the Constant temperature incubator)<br>
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24. Add 500μL of equilibrium liquid BL into the tubes.<br>
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25. Put the tubes into the centrifugal machine to be centrifuged for 1min in 12000 rpm (Remember to arrange the tubes symmetrically).<br>
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26. At the same time of executing 25, take 1.5mL of bacteria liquid and centrifuge in the same situation as 25.<br>
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27. Outwell the effluent and add the turbid liquid (contains RNase and enzyme RNA)<br>
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28. Add P2 and dissociate.<br>
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29. Add P3 and precipitate the protein.<br>
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30. Centrifuge the solutions in 12000rpm for 10 minutes, and absorb the supernatant (contains DNA).<br>
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31. Repeat 30 but change the time to 40-60 seconds.<br>
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32. Outwell the effluent and add anhydrous ethanol, centrifuge the tubes for 1 minute in 12000rpm, outwell the effluent. (Repeat this step for one time)<br>
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33. Elution the solution (add the eluent and wash the tube with buffer).<br>
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34. Measure the concentration of DNA (Do as Part Three)<br>
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※The concentration data we got was 2.07<br>
 +
<br>
 +
Part Five  Digestion(LEAST 3 hours)<br>
 +
35. Choose the buffer (2-3 μL 10X)<br>
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36. Add 14μL DD water.<br>
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37. Add 2μL buffer.<br>
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38. Add 2μL DNA.<br>
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39. Add 2μL enzyme (contained in the cone sugar).<br>
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40. Shake the tubes and put them into the Constant temperature incubator to germiculture under 30 Celsius degree.<br>
 +
--------------------------------------------------------2 hour waiting------------------------------------------------------<br>
 +
<br>
 +
Part Six  Glue Preparation(LEAST 2.5 hours)<br>
 +
41. Decide the size of Gum care and Comb.<br>
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42. Weigh the agarose. (The proportion can be 1.0% or 1.5%, depends on the experimental need and the size of glue).<br>
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43. Solute the agarose with TAE.<br>
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44. Heat the solution and cool it before using.<br>
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45. Pour the solution into the Gum care and cool for 30 minutes.<br>
 +
---------------------------------------------------30 minutes waiting----------------------------------------------------<br>
 +
46. Put the Gum care into the electrophoresis tank.<br>
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47. Drop the colored DNA into the groove on the glue and switch on the electricity. (Remember to put the glue on the side of cathode.)<br>
 +
---------------------------------------------------30 minutes waiting----------------------------------------------------<br><br>
 +
48. Color the glue with EB (EB is REAAAAAALLY DANGEROUS, be careful!)<br>
 +
49. Scan the glue.<br>
 +
<br>
 +
Part Seven  recycle and extract DNA(LEAST 3 hours)<br>
 +
50.follow the kit protocol on the book.<br>
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<br>
 +
Part Eight  Linking(LEAST 3 hours)<br>
 +
51. Get the EP tubes and make marks on them.(Better the name of DNA)<br>
 +
52. Get the glue made in Part Six and put it under the UV-light(remember to wear the safety goggles).<br>
 +
※2I: 1.110<br>
 +
GFP: 1.082<br>
 +
18C: 1,.080<br>
 +
GP: 1.086<br>
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53. Cut the glue with the parts we need.<br>
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※The weight<br>
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Total:<br>
 +
18C: 1.370<br>
 +
GFP: 1.230<br>
 +
2I: 1.244<br>
 +
6P: 1.345<br>
 +
Glue:<br>
 +
18C: 290mg*3 870<br>
 +
GFP: 148mg*3 444<br>
 +
2I: 134mg*3 402<br>
 +
6P: 259mg*3 777<br>
 +
※the weight of glue: solvent should be 1:3, the solvent is Buffer QG<br>
 +
54. Shake the tubes every 2-3 minutes for the duration of 10 minutes under the temperature of 50-60 Celsius degree in order to help dissolving.<br>
 +
55. Add isopropanol (as heavy as the glue), transfer the solution into the tubes, centrifuge for 1 minute, outwell the effluent.<br>
 +
56. Add PE, centrifuge for 1 minute, outwell the effluent.<br>
 +
57. Centrifuge for 1 minute again.<br>
 +
58. Add 50 μL DD water into the tubes.<br>
 +
59. Centrifuge the tubes again.<br>
 +
60. Get the water and DNA ligase.<br>
 +
※10μL in total<br>
 +
GFP: 2μL, buffer 1μL, enzyme 1μL<br>
 +
6P: 2μL, buffer 1μL, enzyme 1μL<br>
 +
2I: 2μL, buffer 1μL, enzyme 1μL<br>
 +
18C: 2μL, buffer 1μL, enzyme 1μL<br>
 +
Polish the solution with DD water.<br>
 +
61. Add those elements showed above(enzyme at last).<br>
 +
--------------------------------------------------------1 hour waiting------------------------------------------------------<br>
 +
Part Nine    Transferring.(the same as Part One,LEAST 2+12 hours)<br>
 +
62. Get the competent cell from the -73C fridge with the commending of our mentors.<br>
 +
63. Draw the DNA solution into the tube of E.coli (by trace extraction) 2μL out of 10μL, and put it inside the ice box for 30 minutes.<br>
 +
--------------------------------------------------30 minutes waiting----------------------------------------------------<br>
 +
64. Turn on the Thermomixer comfort 10min before using.<br>
 +
65. Put E.coli tube into Thermomixer comfort for 42 Celsius degree and 90 seconds “Hot Shock”.<br>
 +
-------------------------------------------------90 seconds waiting------------------------------------------------------<br>
 +
66. Put E.coli back into the ice-box for 5 minutes.<br>
 +
---------------------------------------------------5 minutes waiting-----------------------------------------------------<br>
 +
67. Add 0.5mL LB (Luria-Bertani medium) into E.coli tube.<br>
 +
68. Get 4 culture medium after the tubes were shaken in Thermostatic oscillation incubator.<br>
 +
69. Use spreader to put E.coli on the plate (inside super clean bench and remember to grill the spreader on the alcohol burner before using).<br>
 +
70. Take the plates with E.coli outside and start germiculturing them in the Constant temperature incubator (37 Celsius degree).<br>
 +
<br>
 +
Part Ten  Check if we success or not<br>
 +
71. Take pictures, collect and arrange data, summary.<br>
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Latest revision as of 05:21, 22 June 2013

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