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

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Revision as of 03:36, 17 June 2012

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.