Turn on the super clean table, place the centrifugal tube plate, test tube rack and maker pen, and conduct UV irradiation for 30 minutes.
Prepare 2 ml Eppendorf tubes, PCR tubes, and 30 ml centrifuge tubes, put them into aluminum boxes, and wrap them with gauze. Fill large (1 ml), medium (200 μl), small (20 μl), and 5 ml, 2 boxes of pipette each type, wrap the shell with kraft paper, and bind the pipette box with rubber band. Prepare ddH2O, 0.1 M CaCl2, 15% 0.1 M CaCl2. Prepare 200 ml LB medium, sub pack 40 test tubes, plug the mouth of the test tubes with test tube plugs, bundle 7 test tubes into one, and wrap the mouth of the test tubes with kraft paper. Prepare 50 mlx2 bottles of LB liquid medium. The above items are sterilized at 121 ℃ for 20 minutes.
1. Diluting Primer
Centrifuge the primer dry powder, add the amount of water marked on the tube wall, and obtain the mother liquor (100 mm), which is diluted 10 times with ddH2O.
2. Prepare Amp antibiotic mother liquor (100 mg/ml):
accurately weigh 1g Amp powder in a glass, add 10 ml dH2O to dissolve it, filter it into a sterile 2 ml EP tube with a filter, and store it at minus 20 ℃.
3. Activate 3-tube DH5 α/ Psb1a3 MRFP, 3 tube DH5 α/ Pmd18t insert, 1 tube BL21:
Open the super clean table, ventilate for 10 minutes, spray 70% alcohol on the sterilized LB test tube, amp mother liquor, medium gun head and strain preservation glycerol tube, and put it into the table. Light the alcohol lamp, burn the tube mouth and tube plug, and connect 100 μl to each tube preserved bacterial solution (2.5 μl needs to be added Amp antibiotic mother liquor), and then burn the tube mouth and tube plug again. Plug the tube mouth, bind the tube, and place it in a shaking table for overnight culture (150 rpm, 37 ℃).
1. Plasmid extraction
1. Column balance: add 500 μl balance liquid BL to the adsorption column CP3 (the adsorption column is put into the collection tube), centrifuged at 12000 rpm for 1 minute, poured out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
2. Take 1-5 ml of bacteria cultured overnight, add it to the centrifuge tube, use a Bench Centrifuge, centrifuge at 12000 rpm for 1 minute, and try to suck out the supernatant (when there is more bacterial liquid, the bacterial precipitation can be collected into a centrifuge tube by multiple centrifugations).
3. Add 250 μl solution P1 to the centrifuge tube with bacterial precipitation (please check whether RNase A has been added first), and use a pipette to completely suspend bacterial precipitation.
4. Add 250 μl solution P2 to the centrifuge tube, gently flip up and down 6-8 times make bacteria sufficient cracking.
5. Add 350 μl solution P3 to the centrifuge tube, flip it up and down gently for 6-8 times immediately and mix it well. At this time, white flocculent precipitation will appear. Then 12,000 rpm centrifuge for 10 minutes.
6. Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is put into the collection tube), and pay attention not to suck out the sediment as much as possible. Centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.
7. Add 600 μl rinse solution PW to adsorption column CP3 (please check whether absolute ethanol has been added first), centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.
8. Repeat step 7.
9. Put the adsorption column CP3 into the collection pipe and centrifuge it at 12000 rpm for 2 minutes in order to remove the residual rinsing liquid in the adsorption column.
10. Place the adsorption column CP3 in a clean centrifuge tube and add 50 μl ddH2O drops to the middle of the adsorption membrane, place at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes to collect the plasmid solution into the centrifuge tube.
Solution
2. Prepare 50 ml of 1% agarose gel:
Insert the comb of 2 μl into the 50 ml dispenser. Dissolve 0.5 g agarose in 50 ml Tae buffer, and use alcohol lamp to dissolve agarose. Cool down (based on the standard of not being hot), add 5 μl 10 mg/ml EB solution, shake evenly, and pour into the mixing plate. Wait about 40 minutes for solidification.
3. PCR amplification:
Using high fidelity enzyme PrimeSTAR ® HS amplified the target fragment from pSB1A3-insert, 4x50 μl reaction. 50 μl reaction system is as follows:
Buffer 10 μL
primer 1 1 μL
primer 2 1 μL
Template 2 μL
ddH2O 11 μL
PCR amplification procedure:
1. 98℃，5 min
2. The three reactions are carried out for 30 cycles:
1. 98℃ 10s
2. 55℃ 5s
3. 72℃ (1min/kb) 30s
3. 72℃ 8 min
4. stored in a 4-degree refrigerator
4. Double digestion:
The extracted plasmid pSB1A3-mRFP and the amplified target fragment were double digested by EcoRI and Xhoi. 4x50 μl reaction. 50 μl enzyme digestion system is as follows:
DNA 21 μL
EcoRI 2 μL
XhoI 2 μL
Buffer 5 μL
H2O 20
Enzyme digestion at 37 ℃ for 4H.
4. Gel recovery:
1. Column balance: add 500 μl balance liquid BL to the adsorption column CA2 (the adsorption column is put into the collection tube), centrifuged at 12000 rpm for 1 minute, poured out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
2. Cut a single target DNA band from the agarose gel (try to cut off the excess part) and put it into a clean centrifuge tube, and weigh it.
3. Add equal volume solution PN to the rubber block (if the gel weighs 0.1 g and its volume can be regarded as 100 µL, then add 100 µL PN solution), place it in a 50 ℃ water bath, and gently turn the centrifuge tube up and down during this period to ensure that the rubber block is fully dissolved. If there are still undissolved rubber blocks, you can continue to stand for a few minutes or add some sol solution until the rubber blocks are completely dissolved (if the volume of the rubber blocks is too large, you can cut the rubber blocks into pieces in advance).
4. Add the solution obtained in the previous step to an adsorption column CA2 (the adsorption column is put into the collection pipe), place it at room temperature for 2 minutes, centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection pipe, and put the adsorption column CA2 into the collection pipe.
5. Add 600 μL rinse solution PW to the adsorption column CA2 (please check whether absolute ethanol has been added before use), centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CA2 into the collection pipe.
6. Repeat step 5.
7. Put the adsorption column CA2 into the recovery header, centrifuge at 12000 rpm for 2 min, and try to remove the rinsing liquid as much as possible. Place the adsorption column CA2 at room temperature for several minutes and dry it thoroughly to prevent the residual rinsing solution from affecting the next experiment.
8. Put the adsorption column CA2 into a clean centrifuge tube, drop an appropriate amount of elution buffer EB in the middle of the adsorption membrane, and place it at room temperature for 2 minutes. The DNA solution was collected by centrifugation at 12000 rpm for 2 min. Preserve DNA solution at -20 ℃.
5. Activate one tube of E. coli DH5α and E. coli BL21 (for the preparation of competent cells).
1. Preparation
Open the super clean table and put 1 ml pipette, 200 μ L pipette, 5 ml pipette, 2ml and 30 ml centrifuge tube, 2 x 50 ml LB medium, UV irradiation. Place 0.1 M CaCl2 and 15% 0.1 M CaCl2 solution in a 4 ℃ refrigerator for precooling.
2. Prepare DH5 α And BL21 competent cells：
1. Transfer the activated DH5α to 50 ml medium (37 ℃, 150 RPM), and monitor the OD600 value with a spectrophotometer until the OD600 is 0.4-0.6 (use dH2O as the control, about 2h).
2. Divide the bacterial solution into 30 ml centrifuge tubes (25 ml for each tube). Centrifuge 4 degrees precooling. Put 30 ml centrifuge tube into the centrifuge, centrifuge 4000 g for 10 min, discard the supernatant, add 20 ml of 0.1 M CaCl2 solution, and ice bath for 30 min.
3. Divide the competent cells into 2 ml centrifuge tubes on ice, 100 μL for each tube. Mark and store the competent cells in the refrigerator at -80 ℃.
3. Prepare DH5 α And BL21 competent cells：
3. Prepare DH5 α And BL21 competent cells：
*Note: this system is an empirical system, and the best connection carrier / target fragment volume ratio can also be calculated according to the instructions:
T4 DNA ligase 1 μL
5 x DNA ligase buffer 2 μL
Linear plasmid 1 μL
Insert DNA 6 μL
Connect with PCR instrument at 16 ℃ for 1h. The linked products were stored at - 20 ℃.
4. Preparation of antibiotic plate
Prepare LB solid medium (add 4.5 g agar to 300 ml LB liquid medium),121 ℃, high temperature and high pressures sterilization for 15 minutes. After cooling to about 60 ℃, add 150 μ L 100mg/ml amp antibiotic, shake it evenly, and pour it into a disposable Petri dish (about 10 plates can be poured).
5. Preparation of antibiotic plate
Add 10 μL pSB1A3-target segments to 100 μL competent cells, ice bath for 30min, then 42 ℃ water bath for 60s, then ice bath for 5 min, and finally add 1 ml LB liquid medium, culture for 1h to resuscitate (37 ℃, 150 rpm). In the super clean table, take 100 μL resuscitation solution, use sterile coating rod to coat Amp antibiotic plate.
1. Add 2.5 μL amp antibiotics into LB test tube, select 3 tubes of colonies into lb Test tubes, and culture overnight in a shaking table (37 ℃, 150 rpm).
2. Mark 6 DH5α/pSB1A3-target fragment colony. Select half of the colonies as the template, and use Taq enzyme for colony PCR. The primers were VF and VR, and the reaction system and procedure were carried out according to the instructions. Prepare 50 ml agarose gel and use DL 2000 DNA marker for electrophoresis.
3. Pick the DH5α/pSB1A3- target fragment colony that is verified to be correct add to LB liquid medium, add 2.5 μ L amp antibiotics, expanded culture.
Transform engineering vector into expression host E. coli BL21, Verify experiment.
1. Extracted the plasmid was from the bacterial solution that verified the correct DH5α/pSB1A3-target fragment.
2. Transform the pSB1A3-target fragment into the E. coli BL21.
3. Fluorescence microscope verification experiment:
Basic operation steps of fluorescence microscope:
(1) Install UV protective cover.
(2) Turn on the power control box switch of the high-pressure mercury lamp.
(3) Insert the light barrier and interrupt the light path.
(4) Warm up for 5 minutes.
(5) Place the glass slide containing the sample on the stage.
(6) Select the objective lens.
*(in the order of low power first and then high power)
(7) Rotate the rotating disc of the spectroscope assembly and select the required spectroscope assembly:
*( "O" is used to observe the transmitted light; "Wu" is used to observe blue fluorescence (such as dapl); "WB" is used when observing green fluorescence (such as FITC); "WG" is used for observing red fluorescence (such as TRITC).)
(8) Use barrier filter
(Most of which are built into fluorescence microscope at present)
(9) Adjust the focal length through thick and thin screws.
(10) Turn on the computer connected to the microscope, click the digital imaging system software, and collect digital images.
