Team:NEU CHINA/Protocols
Reaction condition：CutSmart Buffer，37℃. Thermal inactivating: 65℃ 20 min
Time saving digestion reaction system:
| Restriction enzyme | 1 μl |
| DNA | 1 μl |
| 10x NEBuffer | 5 μl (1x) |
| Total Volume | 50 μl |
| Reaction Temperature | 37℃ |
| Reaction time | 5-15min |
1.Thaw competent cells on ice.
2. Mix 40μl competent cell with plasmid (10 ng/μl).
3.Sit the mixture on ice for more than 10 min.
4.Heat shock mixture at 42 ℃ for 90s.
5.Take mixture and sit on ice 2 min.
6.Pipet the mixture into 1ml LB broth and culture at 37 ℃ for 15 min-1h with 200~220 rpm.
7.Centrifuge for 5min at 3,000 rpm.
8.Screening of transformed bacteria by corresponding resistant plates.
9.Heat the triangular plates-smearing stick, and leave it cool for more than 4 min.
10. Evenly spread 150μl of bacteria on the plate. let the plate dry, and inverted culture it in 37℃ incubator.
1.Prepare sterilized CaCl2 solution, PCR tubes, 250mL Conical flask and autoclaved LB broth.
2.Inocμlate BL21 and DH5α into10 ml LB broth and culture bacteria at 37 ℃ with 200~220 r.p.m, overnight.
3.Re-inocμlate OD = 0.2 of bacteria (100mL) in 250mL Conical flask with 100 mL LB medium.
4.Cμlture bacteria at 37 ℃ for 1.5h with 200~220 r.p.m to sustain the bacteria to Log phase, which the OD reading is around 0.4~0.6. Then, transfer bacteria to 50mL centrifugal tube.
5.Sit on ice for 10 mins.
6.Centrifuge for 7min at 3,000 r.p.m in 4℃.
7.Discard supernatant, resuspend bacteria with 10 mL ice cold CaCl2 solution.
8.Centrifuge for 5min at 2,500 r.p.m in 4℃.
9.Discard supernatant, resuspend bacteria with 10 mL ice cold CaCl2 solution.
10.Sit on ice for 30 mins.
11.Centrifuge for 5min at 2,500 r.p.m in 4℃, discard supernatant, resuspend bacteria with 2 mL ice cold CaCl2 solution. Aliquot 100 μl competent cells per PCR tube.
12.Frozen PCR tubes with liquid nitrogen and store in -80℃ refrigerator.
1.Add reagents as follow:
| Template | <1μl |
| Primer1 | 2μl |
| Primer2 | 2μl |
| 2x MasterMix | 25μl |
| ddH2O | Up to 50μl |
| Total | 50μl |
Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the tube.
2.Standard PCR Program:
3.The annealing temperature may vary due to the different primer melting temperatures.
1.Select single colonies from LBA(K) medium. Place on a shaker overnight at 37℃.
2.Centrifuge at 3000r.p.m for 5min. Discard the supernatant, and suck out the excess.
3.Add 250μl Solution I. Transfer it into a clean 1.5ml microcentrifuge tube.
4.Add 250μl Solution II, and gently rotate the tube several times to obtain a clear lysate.
5.Add 350μl Solution III, and invert several times until precipitate forms.
6.Centrifuge at 13000r.p.m for 15min, and take 750μl supernatant into a 2ml collection tube.
7.Centrifuge at 13000r.p.m for 1min and discard the underlying liquid.
8.Add 700μl Wash Buffer and centrifuge at 13000r.p.m for 1min twice.
9. Discard the underlying liquid and centrifuge empty tube at 13000r.p.m for 2min. Discard the underlying tube.
10.Add 50μl Elution Buffer, centrifuge at 3,000r.p.m for 1min.
11.Transfer it into a clean 1.5ml microcentrifuge tube. Let seat at room temperature for 1min. Test its concentration. Store plasmid at -20℃.
1. Mix 3g agarose powder with 30ml 1xTAE.
2. Heat in microwave for 1min until clear.
3. Wait for it to cool down to 40-50℃, and add EB 1μl/30ml.
4. Place the glue tank horizontally, slowly pour the glue liquid so that it forms a uniform level of glue surface, then insert the comb at one end.
5. After the gel has solidified, carefully pull up the comb and put the glue into the electrophoresis tank.
6.Add 1×TAE electrophoresis buffer into the tank until the liquid surface covers the rubber surface.
7. Add sample to sample well. (5μl each)
8. Switch on the electrophoresis apparatus and electrophoresis tank, and then switch on the power supply at 110V for about 15min.
1. Select single colonies from LBA(K) medium. (Add the appropriate proportion of antibiotics in a ratio of 1000 to 1into 5mlLB) Place on a 200r.p.m shaker overnight at 37℃.
2.Move to big shaker.
3.Dilute the bacterial solution to OD=0.2, and add the antibiotic.
4.Shake about 4 to 5 hours, until OD is between 0.4 and 0.6.
5.Add IPTG (100:1) and S protein（50:1）and continue shaking.
6.Measure the fluorescence intensity and OD every two hours. (This step can be done directly on the 96-well plate for easy measurement)
7. Transfer to microscope for observation when the fluorescence intensity is strong enough.
8. At the same time, the gradient of IPTG and S protein can be done on the 96-well plate.
(Take IPTG gradient as an example: the total volume was 90μl, and each adjacent well was diluted 10 times and diluted 5 times.)
1.Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.
2.When adequate separation of bands has occurred, carefμlly excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
3.Determine the appropriate volume of the gel slice by weighing it in a clean 15 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL.
4.Add 1 volume XP2 Binding Buffer.
5.Incubate at 50-60℃ for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
6.Insert a HiBind DNA Mini Column in a 2 mL Collection Tube.
7.Add no more than 700 μl DNA/agarose solution from Step 5 to the HiBind DNA Mini Column.
8.Centrifuge at 10,000 xg for 1 minute at room temperature.
9.Discard the filtrate and reuse collection tube.
10.Repeat Steps 7-9 until all of the sample has been transferred to the column.
11.Add 300 μl XP2 Binding Buffer.
12.Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature.
13.Discard the filtrate and reuse collection tube.
14.Add 700 μl SPW Buffer.
15.Centrifuge at maximum speed for 1 minute at room temperature.
16.Discard the filtrate and reuse collection tube.
17.Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
18. Transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
19. Add 15-30 μl Elution Buffer or deionized water directly to the center of the column membrane.
20. Let sit at room temperature for 2 minutes.
21. Centrifuge at maximum speed for 1 minute.
22. Store DNA at -20℃.
1.Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1-5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hours at 37℃ with vigorous shaking (~300 rpm). Use a 10-20 ml culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an endA negative strain of E. coli be used for routine plasmid isolation. Example: of such strains include DH5a and JM109.
2.Centrifuge at 10,000 x g for 1 minute at room temperature.
3.Decant or aspirate and discard the culture media.
4.Add 250 μl Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.
5.Transfer suspension into a new 1.5 ml microcentrifuge tube.
6.Add 250 μl Solution ll. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.
7.Add 350 μl Solution Ⅲ, Immediately invert several times until a floccμlent white precipitate forms.
8.Centrifuge at maximum speed (213,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
9.Insert a HiBind DNA Mini Column into a 2 mL Collection Tube.
10.Transfer the cleared supernatant from Step 8 by CAREFΜLLY aspirating it into the HiBind DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column.
11.Centrifuge at maximum speed for 1 minute.
12.Discard the filtrate and reuse the collection tube.
13.Add 500 μl HBC Buffer.
14.Centrifuge at maximum speed for 1 minute.
15.Discard the filtrate and reuse collection tube.
16.Add 700 μl DNA Wash Buffer.
17.Centrifuge at maximum speed for 1 minute.
18.Discard the filtrate and reuse the collection tube.
19.Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
20.Transfer the HiBind DNA Mini Column to a clean 1.5 ml microcentrifuge tube.
21.Add 30-100 μl Elution Buffer or sterile deionized water directly to the center of the column membrane.
22.Let sit at room temperature for 1 minute.
23.Centrifuge at maximum speed for 1 minute.
24. Store DNA at -20°C.
1.Prepare some PCR tube, mark the order of them and place them on the yellow plate.
2.Add 2μl double distilled water into the PCR tube.
3.Pick up the colony with a white pipette tip and insert it into the PCR tube.
4.Prepare medium and divide it into areas.
5.Take out the pipette tip in the PCR tube and coat the medium.
6.Put the pipette tip into the PCR tube again.
7.Put the PCR tube with the pipette tip in it on the tube holder and heat them 1~2min in the microwave.
8.PCR system
| DNA | 2μl |
| primer | 0.5μl +0.5μl |
| master mix(2X) | 10μl |
| double distilled water | 7μl |
9.PCR process
1.Prepare the LBA medium(1:1000).
2.Put LBA medium into every centrifuge tube.
3.Pick the bacteria with a pipette tip and add them to each centrifuge tube.
4.Culture in shaker at 37°C overnight.
1.Put the bacteria on ice.
2.Load LB medium into a centrifuge tube.
3.Pick 100 μl of bacteria with a pipette tip and add them to the medium.
4.Culture in shaker at 37°C overnight.
1.Select single colony, and inoculate on 3mL or 10mL LB(K) overnight.
2.Culture the bacterial solution overnight and inoculate into 60mL or 200mL(1:20) LB(K) preheated to 37℃.
3.Culture at 37℃ for 30-60min until OD600 reach 0.5-0.7. (OD600 close to 0.6 is better.)
4.Add IPTG to the final concentration of 1mM, and culture for 4-5 hours.
5.Collect the bacterial liquid into the centrifugal tube. Centrifuge at 4000g for 20 minute at 4℃.
6.Discard the supernatant and collect the precipitation.
1.Precipitate the bacteria, and add 4mL non-denatured lysate to the lysate per gram of wet bacterial weight, then re-suspend the bacteria.
2.Add lysozyme to the final concentration of 1mg/mL, mix well, then place on ice for 30min.
3.Ice ultrasonic cracking. (Power 200-300W) 10s each time, 10s each interval, a total of 6 times.
4.If very thick, add RNase A to 10μl/mL and DNaseI to 5μl/mL. Place on ice for 10-15min.
5.Centrifuge at 10000g for 20 to 30 minutes at 4℃. Collect supernatant of bacterial lysate and place on ice.
6.Take 1mL of evenly mixed 50% BeyoGold His-Tag Purification Resin. Centrifuge at 1000g for 10 seconds at 4℃.
7.Discard the storage fluid and add 0.5mL of non-denaturing lysis fluid to balance the gel. Centrifuge at 1000g for 10 seconds at 4℃. Discard the liquid and repeat 1-2 times.
8.Add about 4mL supernatant of bacterial lysate to it. Place on the shaker for 60min at 4℃.
9.Put the mixture of lysate and BeyoGold His-tag Protein into empty column tube.
10.Open the bottom cover, under the action of gravity, the liquid in the column flows out, collect about 20μl washing solution for subsequent analysis.
11.Wash column 5 times, add 0.5 to 1mL non-denaturing solution each time, collect about 20μl washing solution each time for subsequent analysis.
12.Elute the target protein 6 to 10 times, each time with 0.5mL non-denaturing eluent.
13.Collect each eluent into a different centrifugal tube, and the collected eluent is the purified His-tag Protein.
1.Shake bacteria one night in advance (5ml LB: 5ul antibiotics)
2.Shake 8-12h overnight to large shaking. Use LB to dilute the bacterium OD to 0.2
3.Continue culturing until the OD of the bacterium reaches 0.4-0.6
4.Add different concentrations of gradient IPTG and extracted S protein
5.Measure the fluorescence intensity and OD by 96 well culture plate every hour, until 6h-8h.