◇1. Place competent cells in an ice water bath to freeze. Just after the cells are thawed, add the target DNA (competence: plasmid=10:1) to the cells, dial the bottom of the tube with your fingers, and mix gently.
◇2. Place in an ice water bath for 30 minutes without shaking. The purpose is to allow the plasmid to bind to competent cells.
◇3. Heat shock at 42℃ for 60 seconds without shaking. The purpose is to open the cell surface to allow genes to enter.
◇4. Put it in an ice water bath for 2 minutes without shaking. The purpose is to make the surface of competent cells airtight.
◇5. Add 500μL of sterile SOC medium or LB medium to resuscitate and activate the cells.
◇6. Place in a shaker at 37°C, shake and resuscitate at 150-200rpm for 60 minutes.
◇7. After centrifugation at 3000g×1min, the supernatant was discarded, and an appropriate amount of bacterial solution was spread on the resistant LB plate. After the liquid is soaked up, turn the plate upside down.
◇8. Incubate at 37°C for 12 to 16 hours.
Restriction Enzyme Digestion
◇1. Number the clean, dry and sterilized eppendorf tubes (0.5ml preferably), add 1μg of DNA and 2μl of the corresponding restriction endonuclease reaction 10× buffer solution with a micropipette, and then add re-distilled water to make the total volume It is 19μl. Mix the solution in the tube and add 1μl enzyme solution. Flick the tube wall with your fingers to mix the solution. You can also shake it with a microcentrifuge to concentrate the solution at the bottom of the tube.
Note:When using restriction enzymes, minimize the time it takes to leave the refrigerator to avoid reduced activity.
◇2. After mixing the reaction system, place the eppendorf tube on a suitable support (such as inserting on a foam board) and keep it in a 37°C water bath for 2-3 hours to complete the digestion reaction.
◇3. Add 2μl 0.1mol/L EDTA (pH8.0) to each tube, mix well to stop the reaction, and store in the refrigerator for later use.
◇1. Under UV irradiation, cut the agarose gel slice containing the target DNA fragment as quickly as possible. When cutting the gel, try to cut off the excess gel. The DNA should not be exposed to UV light for more than 30 seconds.
◇2. Transfer the gel block with the target fragment to a 1.5ml centrifuge tube (the centrifuge tube is pre-weighed), and weigh the gel block. Approximately estimate its volume.
Note:After the gel is completely dissolved, pay attention to the pH value of the gel-Binding buffer mixture. If the pH is greater than 8, the yield of DNA will be greatly reduced. Observe the color of the mixture. If it is orange or red, add 5μL 5M,pH5.2 sodium acetate to lower its pH.
◇3. Take a clean HiBind DNA Mini column and put it in a clean 2ml collection tube.
◇4. Transfer all the DNA/gel melt obtained in the third step to the chromatographic column. Centrifuge at 10000g×1min at room temperature. Discard the filtrate in the collection tube and put the chromatographic column back into the 2ml collection tube.
◇5. If the volume of DNA/gel melt exceeds 700μL , only 700μL can be transferred to the column at a time, and the rest can continue to repeat step 4 until all the solutions have passed through the column. Each Hibind DNA recovery and purification column has a limit of 25g DNA adsorption capacity. If the expected yield is large, add the samples to the appropriate number of columns.
◇6. Discard the filtrate in the collection tube and put the chromatographic column back into the 2ml collection tube. Pipette 300μL Binding Buffer (XP2) into the column and centrifuge at 10000g×1min at room temperature.
◇7. Discard the filtrate in the collection tube and put the chromatographic column back into the 2ml collection tube. Transfer 700μL SPW Wash buffer (diluted with absolute ethanol) to the column. centrifuge at 10000g×1min at room temperature.
◇8. Repeat to wash the column with 700ul SPW Wash buffer. Centrifuge at 10000g×1min at room temperature.
◇9. Discard the filtrate in the collection tube and put the column back into the 2ml collection tube. Centrifuge at 13000g×2min at room temperature to dry the remaining liquid of the column matrix.
◇10.Put the column in a clean 1.5ml centrifuge tube, add 15~30μL(depending on the expected final product concentration) of Elution Buffer (or TE buffer) to the column matrix, leave it at room temperature for 1 min, and centrifuge at 13000g×1min to elute the DNA.
◇1. Take 3~4.5ml (1.5ml during verification) of sterile bacteria solution (which already contains RNaseA) and transfer it into a 1.5ml centrifuge tube and mark.
◇2. Centrifuge at 10000g×1min.
◇3. Discard the supernatant.
◇4. Add 250μL of Solution Ⅰ and vortex to mix well.
◇5. Add 250μL of Solution Ⅱ, and immediately turn it upside down 3~4 times for cell lysis.
◇6. Let stand for 2min to make the solution clear and there will be wire drawing phenomenon.
◇7. Add 350μL of Solution Ⅲ, gently invert 3~4 times, the purpose is to settle cell debris.
◇8. Centrifuge at 13000g×1min.
◇9. Pipette the supernatant into the blue tube.
◇10. Centrifuge at 10000g×1min.
◇11. Discard the white tube liquid.
◇12. Add 500μL HBC Buffer to wash small proteins.
◇13. Centrifuge at 10000g×1min.
◇14. Discard the white tube liquid.
◇15. Add 700μL DNA Wsah Buffer to wash the plasmid, if washing twice, the effect will be better.
◇16. Centrifuge at 10000g×1min.Discard the white tube liquid.
◇17. Centrifuge at 13000g×2min with nothing added to remove ethanol, discard the white tube, change to 1.5ml centrifuge tube, and mark.
◇18. Add 30-100μL EB (for storage), let it stand for 2 minutes, and the eluate is used to dissolve the plasmid. If it is genetic manipulation, use 30-100μL water instead of EB.
◇19. Centrifuge at 10000g×1min.
BoC-Lysine,IPTG Solution preparation
Microcystin LR (MC-LR) ELISA test kit
◇1. Take out the required slats from the aluminum foil bag that has been equilibrated at room temperature for 20 minutes, and seal the remaining slats with a ziplock bag and return to 4°C.
◇2. Set up standard wells and sample wells, add 50μL of different concentrations of standard to each standard well;
◇3. Add 10μL of the sample to be tested to the sample well, then add 40μL of sample diluent; do not add to the blank well.
◇4. In addition to the blank wells, add 100μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator 60min.
◇5. Discard the liquid, pat dry on absorbent paper, fill each hole with washing liquid, let it stand for 1 min, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing 5 times (the plate can also be washed with a plate washer).
◇6. Add 50μL each of Substrate A and B to each well, and incubate for 15min at 37°C in the dark.
◇7. Add 50μL of stop solution to each well, and measure the OD value of each well at 450nm wavelength within 15min.
STEP 1 EXTRACTION OF DNA FROM A COLONY
●1.1 Start with large (2 mm) colonies. Colonies can be restreaked into a larger patch on a second plate of selective media if they are very slow-growing, or if the first plate is crowded with colonies.
●1.2 For each colony to be screened, pipette 20 ml 20 mM sodium hydroxide into a 0.2-ml thin-walled PCR tube. Using a pipettor tip, scrape up about half of a colony and pipette up and down in the 20 ml sodium hydroxide to disperse the colony in the liquid. Label the colony on the plate so that positive colonies can later be identified.
●1.3 Incubate tubes for 8 min at 100℃ in a PCR machine, with a heated lid.
●1.4 Briefly spin the tubes in a microfuge to collect liquid at the bottoms of the tubes. Mix each tube vigorously using a vortex mixer at top speed for about 15 s.
●1.5 Spin tubes in a microfuge for about 30 s at top speed to pellet the cell debris.
●1.6 Use the supernatant as the template DNA in a PCR reaction, as described in Step 2.
STEP 2 PCR
●2.1 Make a PCR master mix, and keep on ice:
16.25 ml purified water
2.5 ml 10×PCR buffer
2.5 ml dNTP mix
1.25 ml forward primer
1.25 ml reverse primer
0.25 ml 100×Taq
●2.2 Pipette 24 ml of the PCR master mix into 0.2-ml thin-walled PCR tubes. Keep tubes on ice.
●2.3 Pipette 0.5–1 ml of the supernatant from each sample into a PCR tube containing PCR master mix.
●2.4 Use a thermocycler to carry out PCR. Typical reaction conditions are as follows, for a product up to 1 kb in length:
5 min at 95℃
repeat 30 times:
15 s at 95 ℃
15 s at 57 ℃
1 min at 72 ℃
5 min at 72 ℃
STEP 3 VISUALIZATION OF PRODUCT ON AGAROSE GEL
●3.1 Cast the agarose gel mix into a gel tray after boiling in a microwave oven to completely dissolve the agarose. Take care while heating the gel mix not to allow it to boil over. This can be done while the PCR is running. Fill the gel tank with 1×TBE, the buffer in which the gel will be run. Note that ethidium bromide is toxic. Wear gloves when handling the gel and take care to dispose of it properly.
●3.2 Add 2 ml DNA loading dye to 10 ml of each sample. Load onto the gel.Load DNA ladder (as commercially supplied, or about 500 ng DNA in a volume of about 10 ml, containing loading dye) in one lane.
●3.3 Run the gel at ~100 V for 45 min, or until xylene cyanolhas run about three quarters the length of the gel.
●3.4 Use a UV lightbox to visualize the DNA bands on the gel
Protein gel electrophoresis
◇1. Mix the sample with 2×SDS gel loading buffer at a ratio of 1:1 (V/V) and heat at 100°C for 3 minutes to denature the protein;
◇2.Take out the denatured protein and put it on ice immediately. If the sample is viscous, the DNA can be sheared by ultrasound. Treatment with maximum power for 0.5 to 2 minutes should effectively reduce the viscosity of the lysate to a controllable level (note: this step must be performed on ice);
◇3. If there is any precipitate, centrifuge the sample at 10 000g at 4°C for 10 minutes, transfer the supernatant to another tube, and discard the precipitate;
◇4.Calculate the amount of sample needed to detect the target protein by Western blotting. Generally, the detection limit of Western blotting for medium-sized proteins is about 1 to 5ng. On the 0.75mm thick SDS polyacrylamide gel, each lane can be loaded with 100μg without too much;
◇5. Use a glass micro-injector to add samples in order, and pay attention to loading along the bottom of the sample hole, otherwise the sample will easily float away. The sample volume should not be too much or too little, generally 15～20ul is appropriate. Add 1? SDS gel sample buffer to the sample wells that are not loaded;
◇6.Use a syringe to remove the air bubbles at the bottom of the two glass plates, and connect the electrophoresis device to the power supply (the positive electrode is connected to the tank, and the negative electrode is connected to the tank). The voltage applied to the gel is 8v/cm. After that, the voltage can be increased to 15V/cm, continue electrophoresis until the bromophenol blue reaches the bottom of the separation gel (about 4 hours), turn off the power;
◇7.Remove the glass plate from the electrophoresis device, put it into a porcelain dish, use a syringe to suck up several milliliters of electrophoresis buffer, insert the needle between a glass plate and the gel, being careful not to puncture the gel, Fill the glass plate with electrophoresis buffer from left to right to separate the glass plate from the gel. Cut off a corner near the left to mark the location of the gel
◇1. Store the strain in a glycerin tube and inoculate 3mL LB test tube, 37°C, 250rpm, culture for 12hr.
◇2. On the second day, inoculate 1% seed solution to 100mL LB/500mL Erlenmeyer flask, culture on a shaker at 37℃ to OD600=0.827, add IPTG for induction, 30℃, 250rpm, 6hr.
◇3. 6,000*g, 4°C, centrifuge for 10 minutes, and collect the bacteria.
◇4. Add 10mL lysis buffer to resuspend the bacteria.
◇5. Ultrasonic breaking: ultrasonic time 3s, interval time 5s, power 200W, ultrasonic 5min.
◇6. 13,000*g, 4℃, centrifuge for 10min. The supernatant is transferred to a new centrifuge tube, filtered with a 0.22um or 0.45um filter membrane, and the sample supernatant is applied to a Ni-column.
◇7. After the sample supernatant is roughly clean, add wash buffer to wash the impurities. Generally, 10-15 times the column bed volume of Wash buffer is needed. Use Coomassie G250 to check whether the impurities are clean.
◇8. Collect the protein and perform SDS-PAGE to detect the purity of the purified protein
Protein staining and decolorization
◇1. Take the gel and put it in an appropriate amount of Coomassie Brilliant Blue staining solution to ensure that the staining solution can fully cover the gel. Place it on a horizontal shaker or a side swing shaker and shake slowly, and dye for 1 hour or more at room temperature. Usually, the color of the gel is very close to that of the dyeing solution. When the gel is hardly visible in the dyeing solution, it can be considered that the dyeing is sufficient. Dyeing for 2-4 hours or longer will not negatively affect the final dyeing effect.
◇2. Pour out the staining solution.
◇3. Add an appropriate amount of Coomassie Brilliant Blue staining decolorizing solution to ensure that the decolorizing solution can fully cover the gel. Place it on a horizontal shaker or a side swing shaker and shake slowly, and decolorize at room temperature for 4-24 hours. During this period, replace the decolorizing solution 2-4 times, until the blue background is basically removed, and the staining effect of the protein band reaches the expected. Usually protein bands appear after 1-2 hours of decolorization.
◇4. After the decolorization is completed, soak it with ddH20. When the length of the unstained gel is equal, refer to the MARK protein and compare it with the unstained gel. Cut the gel with the required protein components and collect it. Then the protein to be purified is separated from the gel.