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

From 2012hs.igem.org

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<li>6. Pour the liquid in a 0.6 μl centrifuge tube, label and store at -20°C. </li>
<li>6. Pour the liquid in a 0.6 μl centrifuge tube, label and store at -20°C. </li>
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<p><li><b>Preparation of Ca+ Competent Cells</b></li>
<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>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>
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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>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>  
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<p><li>1.Add the correct antibiotic to a test tube with culture medium. </li>
<p><li>1.Add the correct antibiotic to a test tube with culture medium. </li>
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<p><li>1.Add the correct antibiotic to a test tube containing culture medium.</li>
<p><li>1.Add the correct antibiotic to a test tube containing culture medium.</li>
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<li>3.Incubate at 37°C with vigorous and continuous agitation (250 rpm) from 16 to 18 hours.</li></p>
<li>3.Incubate at 37°C with vigorous and continuous agitation (250 rpm) from 16 to 18 hours.</li></p>
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<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>
<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>
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<p><li>1.Take 1ml of mQ water and place it inside a 1.5mL centrifuge tube.</li>
<p><li>1.Take 1ml of mQ water and place it inside a 1.5mL centrifuge tube.</li>
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<a name="Plasmidic DNA characterization"></a>Plasmidic DNA characterization<div class="br2"></div><div class="br2"></div>             
<a name="Plasmidic DNA characterization"></a>Plasmidic DNA characterization<div class="br2"></div><div class="br2"></div>             
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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>
<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>
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<a name="BioBrick Pieces Assembly"></a>BioBrick Pieces Assembly<div class="br2"></div><div class="br2"></div>             
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<p><li>1.Prepare the mix. (Reaction order: mQ water > Buffer > Enzyme). Using the NEB enzymes.</li>
<p><li>1.Prepare the mix. (Reaction order: mQ water > Buffer > Enzyme). Using the NEB enzymes.</li>
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<a name="Genetic parts ligationa>Genetic parts ligation<div class="br2"></div><div class="br2"></div>             
<a name="Genetic parts ligationa>Genetic parts ligation<div class="br2"></div><div class="br2"></div>             
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<p><li>1.Take in account the concentration of every sample that now contain the fragments which will used bind.</li>
<p><li>1.Take in account the concentration of every sample that now contain the fragments which will used bind.</li>

Revision as of 01:02, 17 June 2012

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Team: UANL_Mty-Mexico

Team: CIDEB-UANL Mexico

Wet-lab
Protocols
Use of plates with BioBrick lyophilazed

  • 1. Search in the Part Registry web site for the desired BioBrick and look forward its exact position in the DNA´s plate.
  • 2. Position correctly the lyophilizated DNA´s plate.
  • 3. Using a 10 μl micropipette, take a white tip and drill the aluminum cover in the well where is located the BioBrick piece.
  • 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.
  • 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.)
  • 6. Pour the liquid in a 0.6 μl centrifuge tube, label and store at -20°C.

    • Preparation of competent cells of E.coli and their transformation

  • Preparation of Ca+ Competent Cells
  • 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.
  • 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).
  • 3.Cool the culture in ice for 5 minutes.
  • 4.Centrifuge for 8 minutes at 1700 × g 4° C.
  • 5.Resuspend gently the pill in 20mL of 0.1 M calcium chloride cooled in ice.
  • 6.Centrifuge for 8 minutes at 1700 × g 4° C.
  • 7.Resuspend the pill in 4ml of 0.1 M calcium chloride cooled in ice.
  • 8.Store in ice for a week until its use.

  • Transformation of Ca+ Competent cells
  • 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).
  • 2.Add 2 μl of DNA and mix giving some light hits among the tubes.
  • 3.Stand on ice from 20 to 30 minutes.
  • 4.Give the thermal shock by immersing the centrifuge tubes inside a beaker with water at 42°C for a minute.
  • 5.Put the tubes back in the ice for 2 minutes.
  • 6.Add 200 μl of LB medium and incubate at 37°C from 20 to 30 minutes.
  • 7.Plate in Petri dishes with LB agar and their respective antibiotic and incubate at 37°C all night long.

    • Inoculation in petri dish and in test tube
  • 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.
  • 2.Take it away from the flame and wait until it’s a little bit cooled.
  • 3.Introduce the sterilized handle inside the tube that contains the bacteria and take a drop of the culture.
  • 4.Take it away from the tube and inoculate the plate by stria forming parallel lines, spreading it over the agar.
  • 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.
  • 6.Incubate the plate at 37°C from 18 to 24 hours.

  • Extension on surface (When cultivating a transformation)
  • 1.Take the inoculum using a micropipette and place it in the agar surface.
  • 2.Take the glass bacteriological loop and introduce it in a beaker with absolute alcohol.
  • 3.Put it away and quickly pass it by the flame and cool it a little bit.
  • 4.Place the glass bacteriological loop in the agar without making any contact with the dumped culture.
  • 5.Spread the inoculum in the plate until it’s dry.
  • 6.Incubate the plate at 37°C from 18 to 24 hours.

    • Inoculation in test tube from plate (Pick colonies for cloning)

  • 1.Add the correct antibiotic to a test tube with culture medium.
  • 2.Pick a colony with a bacteriological loop previously sterilized or pick with the point of the micropipette
  • 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).
  • 4.Incubate at 37°C with vigorous and continuous agitation (250rpm) from 16 to 18 hours.

    • Reculture bacteria from test tube to test tube

  • 1.Add the correct antibiotic to a test tube containing culture medium.
  • 2.Take approx. 20 μL of culture containing bacteria and introduce them into a test tube with the new culture medium.
  • 3.Incubate at 37°C with vigorous and continuous agitation (250 rpm) from 16 to 18 hours.

    • Minipreps of plasmidic DNA

  • 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.
  • 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).
  • 3.Leave all at room temperature from 5 to 10 minutes.
  • 4.Add 200 μL of Solution II and mix by inversion. Leave at room temperature from 5 to 10 minutes.
  • 5.Add 200 μL of Solution III and mix by inversion. Leave all the samples in ice for about 10 minutes.
  • 6.Centrifuge at 14,000 rmp for 5 minutes.
  • 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.
  • 8.Incubate at 20°C for 10 minutes (From 10 minutes to 2 hours).
  • 9.Centrifuge at 14,000 rpm for 10 minutes and throw the supernatant away.
  • 10.Add 200 μL of etanol at 70% and give vortex for a few seconds.
  • 11.Centrifuge at 14,000 rpm for 5 minutes and remove the sobrenatant using a tip.
  • 12.Dry the pill at 37°C for 5 minutes in the incubator.
  • 13.Add 20 μL of mQ water with RNAse (10mg/mL) and resuspend with wortex.
  • 14.Run a gel or store at 4°C (DNA Electroforesis in agarose gel).

    • Quantification of DNA by Ultra Violet Spectrophotometry

  • 1.Take 1ml of mQ water and place it inside a 1.5mL centrifuge tube.
  • 2.Add 1 μL of plasmidic DNA samples of E.coli (Dilution 1:1,000).
  • 3.Calibrate the spectrophotometer with a cuvette containing 1mL of distillated water.
  • 4.Transfer the sample of the centrifuge tube to a cell of the spectrophotometer by using a micropipette.
  • 5.Place the cell in the spectrophotometer.
  • 6.Select the DNA or RNA option in the machine.
  • 7.Select the DNA option in the spectrophotometer.
  • 8.Read the absorbance of the sample with the spectrophotometer.
  • 9.Annotate the reading made at 260, 280 and 320 nm, the relation 260/280 and the concentration given by the spectrophotometer.
  • 10.Remove the cell from the spectrophotometer and throw away its containing into a biological wastes container.
  • 11.Wash the cell, first with distillated water and then with ethanol at 100% and put it in a chemical wastes container.
  • 12.Let the cell dry so it can be reused. (It’s recommended to use the same used solution).

    • Plasmidic DNA characterization

  • 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).
  • 2.Distribute the mix in equal parts.
  • 3.Add the sample of DNA to the reactions and give a gently vortex.
  • 4.Incubate the reactions at 37°C in the incubator from 1 to 12 hours.
  • 5.Run the agarose to check the result.

    BioBrick Pieces Assembly

  • 1.Prepare the mix. (Reaction order: mQ water > Buffer > Enzyme). Using the NEB enzymes.
  • 2.Distribute the mix in equal parts.
  • 3.Add the sample of DNA to the reactions and give a gently vortex.
  • 4.Incubate the reactions at 37°C in the incubator from 1 to 12 hours.
  • 5.Run 10 μL in agarose gel.
  • 6.Store 10 μL for its posterior ligation.

  • 1.Take in account the concentration of every sample that now contain the fragments which will used bind.
  • 2.Use the Ligation Calculator to obtain the quantities to separate the ligation MIX with a final volume of 20 μL.
  • 3.Prepare the mix using the quantities given by the calculator in the following order: Agua mQ >Ligation Buffer>Vector/Fragment ratio.
  • 4.Distribute the ligation mix if necessary and add the ligase T4 (NEB ligase)
  • 5.Incubate the reactions at 25°C (room temperature) for an 1 hour or all night long.

    • iGEM High School Division, 2012 edition - CIDEB-UANL_Mexico team

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