Team:Gifu/Note/


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PROTOCOLS



  • Medium
  • Restriction Digests
  • Ligation
  • Transformation
  • Densitometry(Nanodrop)
  • Colony PCR
  • Electrophoresis
  • Miniprep
  • SDS-PAGE


    • Medium

    • LB medium(100mL)
      • Tryptone ------ 1.0g
      Yeast Extract ------ 0.5g
      NaCl ------ 1.0g
      H2O ------ 100mL
      agar ------ 1.5g (in case of agar medium)


    • SOC medium(100mL)
      • Tryptone ------ 2.0g
      Yeast Extract ------ 0.5g
      1M NaCl ------ 1.0mL
      1M KCl ------ 0.25mL
      H2O (up to 100mL)


      Restriction Digests

      Materials

      • Ice and bucket/container
      • DNA to be digested
      • Buffer M (10x)
      • Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
      • Incubator

      Procedure(an example)

      1. Add 47ul of DNA to be digested into a 1.5ml microcentrifuge tube.
      2. Add 5.0ul of10x buffer M.
      3. Add 1.0ul of EcoRI.
      4. Add 1.0ul of SpeI.
      5. There should be a total volume of 50ul. Mix well and spin down briefly.
      6. Incubate the restriction digest at 38C for 30min. We incubate in an incubator.
      7. Run a portion of the digest on a gel (6ul), to check that part length is accurate.

      Ligation

      1. Add digested fragment A.
      2. Add digested fragment B.
      3. Add ligation mixture.
      4. Ligation 16℃ for 30 min.

      Note: Make the amount of fragment A and B equimolar. And make sure that the volume of ligation mixture is equivalent to the total amount of the volume of fragment A and B. we used a constant temperature water bath.

      Transformation

      1. Deal 25ul of competent cells to each 1.5ml tubes.
      2. Add 1ul of each solution made by ligation to each tube.
      3. Close tubes and incubate the cells for 30 min on ice.
      4. Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 seconds.
      5. Incubate the cells on ice for 2 minutes.
      6. Add 225ul of SOC media to each tube.
      7. Incubate cells at 37C for 30 minutes.
      8. Inoculate with each solution of cells to each plate with appropriate antibiotics.
      9. Culture the cells.

      Densitometry(Nanodrop,In case of nucleic acid)

      1.   Activate the device.
      2.   Choose “Nucleic Acid” at main menu.
      3.   Wash a space of a sample with distilled water and then wipe gently the top and bottom of the arm.
      4.   Choose a sample type. There are DNA-50(double-strand), DNA-33(single-strand), RNA.
      5.   Pour distilled water to test a blank and after that wipe the top and bottom of the arm.
      6.   Pour the sample to measure the concentration.

      Colony PCR

      1. Collect a colony from a plate and then suspend it in 100 µL of LB medium (liquid).
      2. Mix the following reagents.
    　　
    sterile water27.5 µL
    10×PCR buffer5 µL
    2 mM dNTP5 µL
    5 pmol/µL Fw primer2.5 µL
    5 pmol/µL　Rv primer2.5 µL
    0.5U/µL Taq polymerase2.5 µL
    total45 µL
  •   Add 5 µL of the suspension to the mixture of reagents.
  •   Do PCR amplification.
  •   Measure the length of the DNA.

    •     Electrophoresis (preparing 200 mL of agarose solution)

    1. Meter 4 g of agarose.
    2. Add 200 mL of 1× buffer into it.
    3. Wrap the neck of flask and then make a hole on the wrap.
    4. Dissolve the agarose with a microwave oven.
    5. Cool it to a suitable temperature.
    6. Pour the agarose solution into a mold with a comb.
    7. Remove bubbles.
    8. After the gel going solid, dislodge the comb carefully.
    9. Transfer the mold into a phoresis tank.
    10. Immerse the mold in 1× buffer.
    11. Mix 5 µL of DNA solution and 1 µL of 3× dye.
    12. Pour the mixture into the well.
    13. Electrophorese at 100V.
    14. Stop the electrophoresis when the band of dye go up to 3/4 of the gel.
    15. Pick out the gel and then stain it with ethidium bromide (0.5 µg/mL) for 20 minutes.
    16. Observe DNA bands with UV transilluminator.

        Miniprep

       SolⅠ:50mM glucose/25mM Tris-HCl/10mM EDTA
       SolⅡ:0.2N NaOH/1%SDS
       SolⅢ:3M acetic acid/5M potassium
       1. Move 1000μL of culture fluid to a 1.5ml tube.
       2. Centrifuge(14,000rpm,room temperature,1minutes) and remove the top layer by decantation.
       3. Add 50μL of SolⅠand mix it by vortex.
       4. Add 100μL of SolⅡ and mix it very slowly and put it gently on ice for 1minute.
       5. Add 75μL of SolⅢ and mix it slowly and put it gently on ice for 5minutes.
       6. Centrifuge(14,000rpm,4℃,5minutes) and move the top layer to a new tube.
       7. Add 225μL of chloroform/phenol(1:1) and mix it .
       8. Centrifuge(14,000rpm,room temperature,10minutes) and move the top layer to a new tube.
       9. Add 225μL of chloroform and mix it.
       10. Centrifuge(14,000rpm,room temperature,5minutes) and move the top layer to a new tube.
       11. Add 225μL of propan-2-ol and mix it and put it gently for 5 minutes.
       12. Centrifuge(14,000rpm,room temperature,10minutes) and remove the top layer.
       13. Add 500μL of 70% ethanol and turn upside-down to clean the inside of the tube.
       14. Centrifuge(14,000rpm,room temperature,5minutes) and remove the top layer completely and dry it by vacuum drying.
       15. Melt the precipitation by adding 20μL of 20μg/mL RNase A/TE buffer.
       16. Incubate it for 30minutes in 37℃.


        SDS-PAGE

       Protein extraction and Sample preparation

    1. Add 50 µL of culture fluid that was cultured overnight to a liquid culture medium, and then cultivate it for a few hours.
    2. When the bacteria are not too many, add a proper quantity of IPTG to the liquid culture medium.
    3. Cultivate it for a few hours and then store it at low temperature.
    4. Pour 1 mL of the liquid culture into a 1.5ml tube. Centrifuge it(13,000 rpm 4°C 15 minutes) and then discard supernatant fluid. Do twice this step.
    5. Add 300 µL of PBS to the deposition and then stir it.
    6. Sonicate the cell suspension with 4 short bursts of 15 sec followed by intervals of 60 sec for cooling.
    7. Centrifuge(13,000rpm, 4°C, 20minutes)and then collect supernatant into another tube.
    8. Add 100 ul of PBS to the deposition. Shake it with a vortex and then collect it.
    9. Pour 8 µL of the supernatant(=cell extract) into a tube, and 8 µL of the precipitation suspension into another tube.
    10. Add 2 µL of 5×loading dye to the tube and then denature it at 90°C with a block incubator.
    11. Centrifuge and adjust the sample.
    12. Apply 10µL of the sample to SDS gel.

       Making a SDS-PAGE gel and Electrophpresis

    1. Mix and shake quickly reagents to adjust the concentration of separation gel.
    2. Construct a plate for electrophoresis.
    3. Pour separation gel into a gap of the plate (until about 2 cm below a comb).
      Note: Wipe a plate for electrophoresis with 70% ethanol.
      Hold a plate for electrophoresis not to spill the gel.
    4. Pour a proper quantity of Milli Q water into a gap of the plate and then incubate for an hour.
    5. Mix and shake quickly reagents for stacking gel (except APS and TEMED).
    6. Slant the gel plate and absorb multistoried Milli Q water.
    7. Add APS and TEMED to mixed reagents for stacking gel (step5).
    8. Fill the gap of the plate with stacking gel and then insert the comb into the gap of the plate.
      Note: Be careful not to mix bubbles in gel.
    9. Take out the plate and gel together after stacking gel coagulates.
    10. Put the plate and gel into a migration tank with the plate toward outside.
    11. Pour 300 µL of electrophoresis buffer into a phoresis tank. Immerse the gel completely.
    12. Apply 10 µL of the sample and 5 µL of a marker.
    13. Electrophorese at 40 mA in the stacking gel and then at 60mA in the separation gel.
    14. Electrophorese until a pigment comes at an appropriate position.
    15. Stop electrophoresis and then carefully take the gel.
      Note: Use tweezers.
    16. Discard the buffer for electrophoresis and then dye the separation gel with CBB.
    17. Wash the gel plate and the electrophoretic tank with neutral detergent and then rinse it steadily.

       Staining with CBB

    1. Put the gel into fixing solution.
    2. Leave the gel to stand with shaking until a band is dyed yellow.
    3. Collect the fixing solution and then put the gel into CBB dyeing liquid.
    4. Wrap it and then heat it until it is just before boiling with a microwave oven.
    5. Remove the wrap carefully and then let vapor out slowly.
    6. Collect the CBB dyeing liquid and then pour deionized water into a container carrying the gel. Put Kim wipe into the container.
    7. Infiltrate deionized water into the gel for several tens of minutes. Transfer waste liquid into a tank.
    8. Take a picture under UV light and then dry the gel and store it.

    RNase processing

    1. Add 9ul of RNA into a 0.2ml microcentrifuge tube
    2. Add 1ul of 10x buffer M
    3. Add 1ul of 20 U/μl Rnase R (exo-ribonuclease)
    4. Incubate at 37℃for 45min


    Reverse transcription (GoScript Reverse Transcription System/promega)

    1. Add 9ul of RNA into a 0.2ml microcentrifuge tube
    2. Add 1ul of 5pmol/ul reverse primer
    3. Incubate at 70℃ for 5min
    4. Mix the following reagents


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