Team:CIDEB-UANL Mexico/Wet-lab/Protocols

             Wet-Lab

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<li>1. Search in the Part Registry web site for the desired BioBrick and look forward its exact position in the DNA´s plate.</li>

<li>2. Position correctly the lyophilizated DNA´s plate. </li>

<li>3. Using a 10 μl micropipette, take a white tip and drill the aluminum cover in the  well where is located the BioBrick piece. </li>

<li>4. Using the same micropipette, throw away the tip, take another one to take 10 μl of mQ water and introduce this tip in the well. </li>

<li>5. Up and down the liquid using the micropipette a couple of times until the DNA is completely resuspended (when dissolving the DNA, it will give to the water a reddish coloration, so, the more reddish the solution means that the resuspension was well done.)</li>

<li>6. Pour the liquid in a 0.6 μl centrifuge tube, label and store at -20°C. </li>

          <a name="Preparation of competent cells of E.coli and their transformation"></a>Preparation of competent cells of E.coli and their transformation

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<p><li><b>Preparation of Ca+ Competent Cells</b></li>

<li>1.Inoculate a DH5T cells colony in 5ml of Luria Bertani medium (LB) without antibiotic. Incubate all night long at 37 Celsius degrees with constantly agitation.</li>

<li>2.Inoculate 1/100 of the volume of these cells to  100ml of LB medium, incubate at 37 Celsius degrees with constantly agitation until reach a DO6000.34!(~5x108!cel/mL).</li>

<li>3.Cool the culture in ice for 5 minutes.</li>

<li>4.Centrifuge for 8 minutes at 1700 × g 4° C.</li>

<li>5.Resuspend gently the pill in 20mL of  0.1 M calcium chloride cooled in ice.</li>

<li>6.Centrifuge for 8 minutes at 1700 × g 4° C.</li>

<li>7.Resuspend the pill in 4ml of 0.1 M calcium chloride cooled in ice.</li>

<li>8.Store in ice for a week until its use.</li></p>

<p><li><b>Transformation of Ca+ Competent cells</b></li>

<li>1.In a 1.5mL centrifuge tube pre-cooled, add 50 μl of competent bacteria. (It’s very important to keep the materials at 4°C).</li>

<li>2.Add 2 μl of DNA and mix giving some light hits among the tubes.</li>

<li>3.Stand on ice from 20 to 30 minutes.</li>

<li>4.Give the thermal shock by immersing the centrifuge tubes inside a beaker with water at 42°C for a minute.</li>

<li>5.Put the tubes back in the ice for 2 minutes.</li>

<li>6.Add 200 μl of LB medium and incubate at 37°C from 20 to 30 minutes.</li>

<li>7.Plate in Petri dishes with LB agar and their respective antibiotic and incubate at 37°C all night long.</li></p>

          <a name="Inoculation in petri dish and in test tube"></a>Inoculation in petri dish and in test tube<div class="br2"></div><div class="br2"></div>             

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<li>1.Take the bacteriological handle and introduce in the flame of the burner until the point of the handle gets red, so it can be sterilized.</li>  

<li>2.Take it away from the flame and wait until it’s a little bit cooled.</li>

<li>3.Introduce the sterilized handle inside the tube that contains the bacteria and take a drop of the culture.</li>

<li>4.Take it away from the tube and inoculate the plate by stria forming parallel lines, spreading it over the agar.</li>  

<li>5.First, inoculating a corner, sterilize the handle again and spread from a corner the bacteria previously inoculate, sterilize again and finish with wide movements.</li>

<li>6.Incubate the plate at 37°C from 18 to 24 hours.</li></p>

<p><li><b>Extension on surface (When cultivating a transformation)</b></li>

<li>1.Take the inoculum using a micropipette and place it in the agar surface.</li>

<li>2.Take the glass bacteriological loop and introduce it in a beaker with absolute alcohol.</li>

<li>3.Put it away and quickly pass it by the flame and cool it a little bit.</li>

<li>4.Place the glass bacteriological loop in the agar without making any contact with the dumped culture.</li>

<li>5.Spread the inoculum in the plate until it’s dry.</li>

<li>6.Incubate the plate at 37°C from 18 to 24 hours.</li></p>

          <a name="Inoculation in test tube from plate (Pick colonies for cloning)"></a>Inoculation in test tube from plate (Pick colonies for cloning)<div class="br2"></div><div class="br2"></div>             

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<p><li>1.Add the correct antibiotic to a test tube with culture medium. </li>

<li>2.Pick a colony with a bacteriological loop previously sterilized or pick with the point of the micropipette</li>

<li>3.Shake by making small circles in the culture (if a handle was used) or up and down the culture a couple of times (if a micropipette was used).</li>

<li>4.Incubate at 37°C with vigorous and continuous agitation (250rpm) from 16 to 18 hours.</li></p>

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          <a name="Reculture bacteria from test tube to test tube"></a>Reculture bacteria from test tube to test tube

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<p><li>1.Add the correct antibiotic to a test tube containing culture medium.</li>

<li>2.Take approx. 20 μL of culture containing bacteria and introduce them into a test tube with the new culture medium.</li>

<li>3.Incubate at 37°C with vigorous and continuous agitation (250 rpm) from 16 to 18 hours.</li></p>

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  <a name="Minipreps of plasmidic DNA"></a>Minipreps of plasmidic DNA <div class="br2"></div><div class="br2"></div>             

<p><li> 1.Add 1.5 mL of culture inside a centrifuge tube. Centrifuge at 14000 rpm for 30 seconds and throw the supernatant away inside a container with chlorine at 0.1% or with liquid soap.</li>

<li>2.Add 200 μL of Solution I and mix giving vortex until the pill is completely dissolved. (A micropipette can be used if it’s difficult to dissolve).</li>

<li>3.Leave all at room temperature from 5 to 10 minutes.</li>

<li>4.Add 200 μL of Solution II and mix by inversion. Leave at room temperature from 5 to 10 minutes.</li>

<li>5.Add 200 μL of Solution III and mix by inversion. Leave all the samples in ice for about 10 minutes.</li>

<li>6.Centrifuge at 14,000 rmp for 5 minutes.</li>

<li>7.Pass the supernatant inside a new centrifuge tube containing 1mL of ethanol at 100% by using a tip, being careful of not passing any precipitate.</li>

<li>8.Incubate at 20°C for 10 minutes (From 10 minutes to 2 hours).</li>

<li>9.Centrifuge at 14,000 rpm for 10 minutes and throw the supernatant away.</li>

<li>10.Add 200 μL of etanol at 70% and give vortex for a few seconds.</li>

<li>11.Centrifuge at 14,000 rpm for 5 minutes and remove the sobrenatant using a tip.</li>

<li>12.Dry the pill at 37°C for 5 minutes in the incubator.</li>

<li>13.Add 20 μL of mQ water with RNAse (10mg/mL) and resuspend with wortex.</li>

<li>14.Run a gel or store at 4°C  (DNA Electroforesis in agarose gel).</li></p>

          <a name="Quantification of DNA by Ultra Violet Spectrophotometry"></a>Quantification of DNA by Ultra Violet Spectrophotometry <div class="br2"></div><div class="br2"></div>             

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<p><li>1.Take 1ml of mQ water and place it inside a 1.5mL centrifuge tube.</li>

<li>2.Add 1 μL of plasmidic DNA samples of E.coli (Dilution 1:1,000).</li>

<li>3.Calibrate the spectrophotometer with a cuvette containing 1mL of distillated water.</li>

<li>4.Transfer the sample of the centrifuge tube to a cell of the spectrophotometer by using a micropipette.</li>

<li>5.Place the cell in the spectrophotometer.</li>

<li>6.Select the DNA or RNA option in the machine.</li>

<li>7.Select the DNA option in the spectrophotometer.</li>

<li>8.Read the absorbance of the sample with the spectrophotometer.</li>

<li>9.Annotate the reading made at 260, 280 and 320 nm, the relation 260/280 and the concentration given by the spectrophotometer.</li>

<li>10.Remove the cell from the spectrophotometer and throw away its containing into a biological wastes container.</li>

<li>11.Wash the cell, first with distillated water and then with ethanol at 100% and put it in a chemical wastes container.</li>

<li>12.Let the cell dry so it can be reused. (It’s recommended to use the same used solution).</li></p>

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<p><li>1.Prepare the digestion mix with the restriction enzymes needed ( Reaction order: mQ water enzyme > Buffer > Enzyme buffer > DNA to get a final volume of 10 μL).</li>

<li>2.Distribute  the mix in equal parts.</li>

<li>3.Add the sample of DNA to the reactions and give a gently vortex.</li>

<li>4.Incubate the reactions at 37°C in the incubator from 1 to 12 hours.

<li>5.Run the agarose to check the result.</p>

<a name="BioBrick Pieces Assembly"></a>BioBrick Pieces Assembly<div class="br2"></div><div class="br2"></div>          

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<a name="Genetic parts ligationa>Genetic parts ligation<div class="br2"></div><div class="br2"></div>          

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     <div class="lateral-button"><a href="https://2011.igem.org/Team:UANL_Mty-Mexico/Modelling/Overview#Overview">Overview</a></div>