Difference between revisions of "Team:Gifu/Note/"

    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

Reverse transcription (GoScript Reverse Transcription System/promega)