Our method
02 Oct,2021
1. PCR system
2. 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.
3. Enzyme digestion
4. Gel electrophoresis
Prepare agarose gel
Experimental supplies: 1×TAE, agarose
1. Weigh agarose according to its working concentration. Usually 0.8% or 1% is suitable for running quantitative gel or testing PCR product.
2. Using 1×TAE to dissolve the agarose by careful warming. (Heat it in microwave oven and boil it two times so that the agarose dissolves completely)
3. When the temperature decreases to 55 degrees Celsius, add Gelstain and shake it up. (1μl Gelstain per 10 ml gel)
4. Pour the prepared gel into the electrophoresis bath, wait for the gel to concrete and reserve it.
Add sample
1. For running quantitative gel, add 1μl sample; For testing PCR product, add 3-5μl sample.
2. Mix up the sample and 10×loading buffer, add the mixture into spotting hole. Be careful not to leave any air bubbles, which will change the volume of the sample.
3. Add corresponding volume of Marker according to the manual. (Usually 5μl Marker per 10μl spotting hole)
4. Set up constant voltage 170V, switch on for about 15 minute. When the indicator line reach 1/2-2/3 of the whole gel, switch off.
5. Use Gel imager to show the imaging of the gel.
5. Agarose Gel DNA Extraction
1. Agarose gel is prepared using TAE buffer or TBE buffer, then agarose gel electrophoresis is performed on the target DNA.
2. Cut the agarose gel containing the target DNA under the UV lamp and suck the liquid on the gel surface with a paper towel. At this point, we should pay attention to the removal of the gel which does not contain the target DNA, minimize the gel volume and increase the DNA recovery rate. When glue blocks exceed 300 mg, use more than one Column Recycling, otherwise seriously affecting the yield. Note: when cutting, please be careful not to expose the DNA to UV light for a long time to prevent DNA damage.
3. Shredding the gel block. When the adhesive block is chopped up, the dissolution time of the block in the operation step 6 can be accelerated, and the DNA recovery rate can be improved.
4. Weighing the weight of the glue block and calculating the volume of the glue block. When calculating the volume of the block, 1mg=1μL is used to calculate.
5. Buffer GM are added into the gel block, and the amount of solution Buffer GM.
6. After mixing evenly at room temperature 15-25 degrees C dissolve glue block (glue concentration is more difficult to dissolve and can be heated at 37 C). At this point should be interrupted oscillation mixing, so that the block fully dissolved (about 5~10 minutes). Note: the block must be fully dissolved, otherwise it will seriously affect the recovery rate of DNA. High concentration gel can prolong the time appropriately.
7. When the gel is completely dissolved, observe the solution color, if the solution color changed from yellow to orange or pink, add 3 M sodium acetate solution to the rubber block solution (pH5.2) 10μL, mixed solution to restore yellow. When the DNA fragment is less than 400bp, the isopropanol with a final concentration of 20% should be added to the solution.
8. Place the Spin Column in the reagent kit on the Collection Tube.
9. Transfer the solution of the operation step 7 to the Spin Column with 12000 rpm centrifugation for 1 minutes, filtrate rejection. Note: if the filtrate is added to the Spin Column to be centrifuged once, it can be improved DNA recovery rate.
10. Add 700μL Buffer WB to Spin Column at room temperature 12000 rpm centrifuge for 30 seconds, discard filtrate. Note: please confirm that Buffer WB has added 100% ethanol of the specified volume.
11. Repeat step 10.
12. Place the Spin Column on the Collection Tube at room temperature 12000rpm Centrifuge for 1 minutes.
13. Place the Spin Column on the new 1.5 ml centrifuge tube at Spin. Add 30μl of sterile water or Elution Buffer at the center of the Column film and leave it for 1 minutes at room temperature. Note: heating the sterilized water or Elution Buffer to 60 degrees can improve the elution efficiency when used.
14. 12000 rpm centrifugations at room temperature, 1 minute elution DNA.
6. Preservation in Glycerol
1. The manufacture of glycerin tubes
Configuration 50% glycerin (Glycerol: ddH2O = 1:1) Packaged in 2ml storage tubes and 550μl per tube Sterilization at 121 degrees for 20min
2. Preservation
In advance, the target strains were cultured in the liquid medium containing antibiotics (E. coli in LB medium, yeast in YPD) for 12-14h Secondly, the liquid fraction of 10ml~20ml was centrifuged twice, and the bacterial precipitate was suspended by 1ml without the corresponding liquid medium of μ antibiotics. Add the suspension to the sterilized glycerin tube, blow it several times and screw down the lid.
7. Electric transfer
the cleaning of the electric rotor: Clean with distilled water, immerse with alcohol in the clean bench for 10min, add sterile water, use a syringe suction cleaning for 3 times, absorb liquid and open the cover (lid should be washed) under UV for 1H, place in ice precooling.
1. Add 5~10μg DNA volume (less tan 10μL) to the competent cell, blow with a gun (gently); (9 L PYD1, 1- alpha 1 g/ L), transfer to the pre-cooled electric cup, and put aside for 5min;
2. Set shock parameters: 1.5Kv, 25 F, 200Ω;
3. Immediately add 1ml precooling sorbitol, transfer to the EP tube, 30 degrees and be static for 1h;
4. 4000rpm and 5min are set to centrifuge at normal temperature, and then supernatant is abandoned and add 1ml YPD, centrifuge at 30 degrees for 200rpm and 2h;
5. 4000rpm, 5min at room temperature centrifugation, suction 550μL supernatant, 150μL/ plate used for coating board [before coating board, culture board should be drying.
8. SDS-PAGE
1. The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.
2. After ensuring that the equipment is waterproof, the 12% (or 18%) running gel is mixed and filled into the chamber. Pipetting about 1 ml of H2O on top of the running gel to seal the gel.
3. After polymerization, the remaining H2O is removed and the 12% stacking gel is filled on top. Insert a comb to create sample pockets.
4. After the stacking gel also polymerized, 1x running buffer is used to run the Double Gel System via the SDS gel.
5. After loading the generated pockets with the samples, the stacking gel is run at 100 V and then running gel at 120 V.
