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 PCR2. Use the PCR product of step 1 as template to do PCR3. 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
2) Enzyme digestion to get linear plasmid (50μl system)
X: look up the enzyme digestion table
y: 2.5μg
5. Infusion
1) Primer design
1. Each In-Fusion primer must contain two parts: the primer 5 'end must be included and will be connected to the DNA fragment (such as carrier or another fragment) 15 ends completely homologous base. Primers 3 must contain primer sequences of target genes.
2. Each primer 3' should be:
- with gene specificity
- length is 18 - 25, the content of GC is 40 - 60%
- annealing temperature (Tm) is of 58 - 65 C. Forward and reverse primers of the Tm value difference should not exceed 4 degrees, otherwise the amplification effect is not ideal. Note that the evaluation of Tm values should be based on primer 3 (gene specificity), rather than on the whole primer. If Tm is too low, we can appropriately extend the part of primer with gene specificity until Tm reaches 58 - 65 degrees C. - do not contain successive identical nucleotides. The last 5 nucleotides of each primer '3' should not contain more than 2 guanine (G) or cytosine (C).
3. The complementary sequence between the primers is avoided to prevent the primer itself from producing hairpin structure.
4. You can do BLAST search to make sure that the 3’ end of each primer is highly specific. (www.ncbi.nlm.nih.gov/BLAST/)
2) Reaction system and steps
* linear plasmid and target gene volume should be smaller than 2μL
1. mixing system
2. static in 50 degrees Celsius environment, reaction for 15min
3. convert on ice, or -20 preservation
4. 2.5μL reaction liquid was added into 50μL competent cells during transformation
6. DNA fragment purification
1. Add Buffer DC as much as 3 times the volume of the PCR reaction liquid to the PCR reaction liquid (or other enzymatic reaction fluid), then mix them up. (If the needed Buffer DC is less than 100μl, then add to 100μl.)
2. Place the Spin Column on the Collection Tube.
3. Transfer the solution in Step (1) into the Spin Column, then centrifuge it at 12000rpm, under room temperature for 1 minutes and throw away the filtrate. (If the filtrate is added back to the Spin Column to centrifuge again, the recovery rate of the DNA can be improved.)
4. Add 700μl Buffer WB to the Spin Column then centrifuge it at 12000rpm, under room temperature for 30 seconds and throw away the filtrate. (Make sure that specified volume of 100% ethanol has been added to the Buffer WB.)
5. Repeat Step (4) once.
6. Place the Spin Column on the Collection Tube, then centrifuge it at 12000rpm, under room temperature for 1 minutes.
7. Place the Spin Column on a new 1.5ml centrifuge tube. Add 25-30μl 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.)
8. Centrifuge the solution at 12000rpm, under room temperature for 1 minutes to elute DNA.
7. Gel electrophoresis
1) 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.2) 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.8. 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.