Difference between revisions of "Team:BNU-China/Protocol"

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                     <p>1. Culture of E-coli: Select single colonies from plate culture medium then inoculate them into 1-3ml liquid nutrient medium containing antibiotics, then culture them at 37 degrees Celsius overnight. (Usually the culture time is 12-16 hours, because if the cells have been cultured over 16 hours, it is hard to lysis the cells so that plasmid production will decrease. Besides, the medium should not be excessive, because too many bacteria are difficult to be fully lysised so that plasmid purity is affected.)</p>
 
                     <p>1. Culture of E-coli: Select single colonies from plate culture medium then inoculate them into 1-3ml liquid nutrient medium containing antibiotics, then culture them at 37 degrees Celsius overnight. (Usually the culture time is 12-16 hours, because if the cells have been cultured over 16 hours, it is hard to lysis the cells so that plasmid production will decrease. Besides, the medium should not be excessive, because too many bacteria are difficult to be fully lysised so that plasmid purity is affected.)</p>
 
                     <p>2. Collect 1-4ml overnight culture of bacterial fluid, then centrifuge it at 12000rpm for 2 minutes and throw away the filtrate.</p><p>3. Use 250μl Solution | (be sure that RNase A is added to Solution |) to fully suspend the bacterial precipitation. (Pay attention not to leave over small pieces of bacteria. Vortex can be used to make the bacteria fully suspended)</p><p> 4. Add 250μl Solution || and gently blend the solution by turning it up and down for 5-6 times to make the bacteria fully lysis until the solution becomes transparent. (Be sure to blend the solution gently instead of oscillating fiercely, and this step should be no more than 5 minutes.)</p><p>5. Add 350μl Solution ||| which was pre-cooled at 4 degrees Celsius, gently blend the solution by turning it up and down for 5-6 times until tight agglutination block is formed, then stand it under room temperature for 2 minutes.</p><p>6. Centrifuge the solution at 12000rpm for 10 minutes and keep the filtrate. (Centrifuge at 4 degrees Celsius is not benefit to sedimentation.)</p><p>7. Place the Spin Column on the Collection Tube.</p><p>8. Transfer the supernatant from Step (6) to the Spin Column, then centrifuge it at 12000rpm for 1 minutes and throw away the filtrate.</p><p>9. Add 500μl Buffer WA to the Spin Column then centrifuge it at 12000rpm for 30 seconds and throw away the filtrate.</p><p>10. Add 700μl Buffer WB to the Spin Column then centrifuge it at 12000rpm for 30 seconds and throw away the filtrate. (Make sure that specified volume of 100% ethanol has been added to the Buffer WB.)</p><p>11. Repeat Step (10) once.</p><p>12. Place the Spin Column on the Collection Tube, then centrifuge it at 12000rpm, under room temperature for 1 minutes and throw away the filtrate.</p><p>13. Place the Spin Column on a new 1.5ml centrifuge tube. Add 50μl sterile water or Elution Buffer to the center of the membrane of the Spin Column and stand the solution under room temperature for 1 minutes. (If the sterile water or Elution Buffer is heated to 60 degrees Celsius, then the elution efficiency can be improved.)</p><p>14. Centrifuge the solution at 12000rpm, under room temperature for 1 minutes to elute DNA.</p>
 
                     <p>2. Collect 1-4ml overnight culture of bacterial fluid, then centrifuge it at 12000rpm for 2 minutes and throw away the filtrate.</p><p>3. Use 250μl Solution | (be sure that RNase A is added to Solution |) to fully suspend the bacterial precipitation. (Pay attention not to leave over small pieces of bacteria. Vortex can be used to make the bacteria fully suspended)</p><p> 4. Add 250μl Solution || and gently blend the solution by turning it up and down for 5-6 times to make the bacteria fully lysis until the solution becomes transparent. (Be sure to blend the solution gently instead of oscillating fiercely, and this step should be no more than 5 minutes.)</p><p>5. Add 350μl Solution ||| which was pre-cooled at 4 degrees Celsius, gently blend the solution by turning it up and down for 5-6 times until tight agglutination block is formed, then stand it under room temperature for 2 minutes.</p><p>6. Centrifuge the solution at 12000rpm for 10 minutes and keep the filtrate. (Centrifuge at 4 degrees Celsius is not benefit to sedimentation.)</p><p>7. Place the Spin Column on the Collection Tube.</p><p>8. Transfer the supernatant from Step (6) to the Spin Column, then centrifuge it at 12000rpm for 1 minutes and throw away the filtrate.</p><p>9. Add 500μl Buffer WA to the Spin Column then centrifuge it at 12000rpm for 30 seconds and throw away the filtrate.</p><p>10. Add 700μl Buffer WB to the Spin Column then centrifuge it at 12000rpm for 30 seconds and throw away the filtrate. (Make sure that specified volume of 100% ethanol has been added to the Buffer WB.)</p><p>11. Repeat Step (10) once.</p><p>12. Place the Spin Column on the Collection Tube, then centrifuge it at 12000rpm, under room temperature for 1 minutes and throw away the filtrate.</p><p>13. Place the Spin Column on a new 1.5ml centrifuge tube. Add 50μl sterile water or Elution Buffer to the center of the membrane of the Spin Column and stand the solution under room temperature for 1 minutes. (If the sterile water or Elution Buffer is heated to 60 degrees Celsius, then the elution efficiency can be improved.)</p><p>14. Centrifuge the solution at 12000rpm, under room temperature for 1 minutes to elute DNA.</p>
 +
                    <h2>4. Enzyme digestion</h2>
 +
                    <h3>1)  Enzyme digestion test (10μl system)</h3>
 +
                    <figure class="text-center">
 +
                        <img src="https://static.igem.org/mediawiki/2018/b/b5/T--BNU-China--image_p5.jpg" width="100%">
 +
                        <figcaption>
 +
                        </figcaption>
 +
                    </figure>
 +
                    <p>X: look up the enzyme digestion table<br>
 +
Y: If the length of the minimal fragment is a, the length of the plasmid is b, the concentration of the plasmid is c, then: 20ng/a=(m ng)/b; y=m/c</p>
 +
 
                      
 
                      
 
                      
 
                      

Revision as of 18:27, 17 October 2018

Team:BNU-CHINA - 2016.igem.org style = "font-family: Helvetica;"

Protocol

1. PCR system

1) 20μL system is used for verification

Taq:

Super mix:

2) 50μL system for PCR target genes

Pfu mix:

DNA: plasmid 50ng; genome 100~200ng

3) 20μL PCR system for microbes

Super mix:

Bacteria (after picking on the board, back up on the new board and rinse it in the system that has been prepared)

4) Fusion PCR

1. Basic PCR
2. Use the PCR product of step 1 as template to do PCR
3. Use the PCR product of step 2 as template to do PCR, but first five cycles don't add primer, add primer at the sixth cycle and continue PCR.

The system of step 2:
H2O 21μL
2x primeSTAR 25μL
R+F-Primer 2 μL
Template1 1μL
Template2 1μL

2. Plasmid concentration

1. Dilute collected DNA to 400μl. (If the plasmid is eluted by ddH2O, then it is diluted by ddH2O as well; If the plasmid is eluted by elution buffer, then it is diluted by elution buffer as well)

2. Add 900μl absolute ethanol to precipitate DNA, and 40μl NaAc (3M PH=5.2) to help precipitation. Be sure to mix it up.

3. Put the reactant in ice-bath for 1 hour, or put it under -20 degrees Celsius overnight.

4. Centrifuge the reactant under 12000rpm for 10 minutes, then throw away the supernatant.

5. Add 1ml 75% ethanol (the 75% ethanol is prepared by using absolute ethanol rather than ethanol for disinfection) and mix up to wash away salt ions.

6. Centrifuge the reactant under 12000rpm for 5 minutes, then throw away the supernatant.

7. Centrifuge the reactant for another 3-4 seconds and use transfer liquid gun to throw away residual supernatant.

8. Centrifuge the reactant for another 3-4 seconds and use transfer liquid gun to throw away residual supernatant.

9. Open the centrifuge tube and expose the reactant to air. Put the centrifuge tube into incubator at 37 degrees Celsius for 15 minutes until the plasmid turn into transparent.

10. Add appropriate volume of ddH2O to dilute the plasmid according to precipitation capacity.

11. Use 1μl solution and add 9μl ddH2O to dilute it, then use electrophoresis to measure its quantity.

3. Plasmid purification

1. Culture of E-coli: Select single colonies from plate culture medium then inoculate them into 1-3ml liquid nutrient medium containing antibiotics, then culture them at 37 degrees Celsius overnight. (Usually the culture time is 12-16 hours, because if the cells have been cultured over 16 hours, it is hard to lysis the cells so that plasmid production will decrease. Besides, the medium should not be excessive, because too many bacteria are difficult to be fully lysised so that plasmid purity is affected.)

2. Collect 1-4ml overnight culture of bacterial fluid, then centrifuge it at 12000rpm for 2 minutes and throw away the filtrate.

3. Use 250μl Solution | (be sure that RNase A is added to Solution |) to fully suspend the bacterial precipitation. (Pay attention not to leave over small pieces of bacteria. Vortex can be used to make the bacteria fully suspended)

4. Add 250μl Solution || and gently blend the solution by turning it up and down for 5-6 times to make the bacteria fully lysis until the solution becomes transparent. (Be sure to blend the solution gently instead of oscillating fiercely, and this step should be no more than 5 minutes.)

5. Add 350μl Solution ||| which was pre-cooled at 4 degrees Celsius, gently blend the solution by turning it up and down for 5-6 times until tight agglutination block is formed, then stand it under room temperature for 2 minutes.

6. Centrifuge the solution at 12000rpm for 10 minutes and keep the filtrate. (Centrifuge at 4 degrees Celsius is not benefit to sedimentation.)

7. Place the Spin Column on the Collection Tube.

8. Transfer the supernatant from Step (6) to the Spin Column, then centrifuge it at 12000rpm for 1 minutes and throw away the filtrate.

9. Add 500μl Buffer WA to the Spin Column then centrifuge it at 12000rpm for 30 seconds and throw away the filtrate.

10. Add 700μl Buffer WB to the Spin Column then centrifuge it at 12000rpm for 30 seconds and throw away the filtrate. (Make sure that specified volume of 100% ethanol has been added to the Buffer WB.)

11. Repeat Step (10) once.

12. Place the Spin Column on the Collection Tube, then centrifuge it at 12000rpm, under room temperature for 1 minutes and throw away the filtrate.

13. Place the Spin Column on a new 1.5ml centrifuge tube. Add 50μl sterile water or Elution Buffer to the center of the membrane of the Spin Column and stand the solution under room temperature for 1 minutes. (If the sterile water or Elution Buffer is heated to 60 degrees Celsius, then the elution efficiency can be improved.)

14. Centrifuge the solution at 12000rpm, under room temperature for 1 minutes to elute DNA.

4. Enzyme digestion

1) Enzyme digestion test (10μl system)

X: look up the enzyme digestion table
Y: If the length of the minimal fragment is a, the length of the plasmid is b, the concentration of the plasmid is c, then: 20ng/a=(m ng)/b; y=m/c