Team:ECUST/Protocol
Basic techniques in Molecular biology
Polymerase chain reaction
| Total volume | 50/μL |
|---|---|
| DDW | 36 |
| 10×Buffer | 5 |
| dNTP（2.5mM） | 5 |
| FP（10μM） | 1 |
| RP（10μM） | 1 |
| DNA template | 1 |
| EasyPfu DNA Polymerase | 1 |
Thermocycling
The PCRmachine should be set to run the following steps
| Pre-denaturation | 94℃ | 5min | |
|---|---|---|---|
| transsexual | 94℃ | 30s | 30circles |
| annealing | Tm-5℃ | 30s | |
| extend | 72℃ | 0.5kb/min | |
| extend | 72℃ | 5min | |
extraction plasmid
1-4 mL of the bacterial solution is centrifuged at 12,000 rpm for 2 min, and the bacterial solution is discarded.
2) Resuspend by adding 200μL SolutionI, then add 400μL SolutionII and invert upside and down slowly until the bacterial solution is transparent. Next add 300μL SolutionIII and mix. Further, 300 ul of chloroform is added and mixed, and centrifuge at 13,000 rpm for 2 minutes. 700 ul of the supernatant is removed into a new 1.5 mL tube, and add 490 μL of 70% isopropanol.Next centrifuge at 13,000 rpm for 2 minutes, and the supernatant is discarded. After rinsing the precipitate twice with 500 μL of 75% ethanol, centrifuges at 12,000 rpm for 2 min.Finally,dry at 65℃ and dissolve with 15-20 mL of sterile water previously heated.
PCR Purfication and gel extraction
PCR Purfication
Add 3 times volume of Buffer PCR-A to the PCR reaction solution; mix and transfer to the preparation tube.Place the preparation tube in a 2 ml centrifuge tube, centrifuge at 12000*g for 1 min and discard the filtrate.
Place the preparation tube back into a 2 ml centrifuge tube, add 700 μL Buffer W2, centrifuge at 12000*g for 1 min, and discard the filtrate.
Place the preparation tube back in a 2 ml centrifuge tube, add 400 μL Buffer W2, centrifuge at 12000*g for 1 min, and discard the filtrate.
Reset the preparation tube to the centrifuge and centrifuge at 12000*g for 1 min.
The preparation tube is placed at 65℃ to dry to an ethanol-free odor, and the preparation tube is placed in a clean 1.5 ml centrifuge tube, and 25-30 μL of Eluent or sterile water is added to the center of the preparation tube, and allow to stand at room temperature for 1 min. The DNA is eluted by centrifugation at 12000*g for 1 min.
gel extraction
The enzymatically digested product is electrophoresed, and the gel containing the target fragment was cut out under a gel imager. The GK2041 gel recovery kit was used for gel recovery. The specific steps are as follows:
(1) Tapping
(2) Add 400ul Binding Solution and bath at 55°C for 4min
(3) Transfer the above mixture to the adsorption column GC-3u with a 2 ml collection tube, place it at room temperature for 2 min, centrifuge at 6000 rpm for 1 min at room temperature, and drain the waste liquid from the collection tube.
(4) Put the adsorption column back into the collection tube, add 500 μL WA Solution, centrifuge at 12000 rpm for 1 min at room temperature, and drain the waste liquid.
(5) Resorb the column again, add 500 μL Wash Solution, centrifuge at 12000 rpm for 1 min at room temperature, and drain the waste.
(6) Repeat (5)
(7) The adsorption column is put back again, centrifuge at 12000 rpm for 1 min, then open the lid at room temperature for 10 min.
(8) Place the adsorption column into a clean 1.5ml collection tube, add 25μL Elution Buffer to the center of the membrane, cover the lid, place at 37℃ for 2min, and centrifuge at 12000rpm for 1min.
agarose gel electrophoresis
Agarose gel electrophoresis was used to separat DNA fragments. For fragments with a size of more than 500 bp, 1% (m/V) agarose gels were used. For the production of gels, the appropriate amount of agarose was solved in 50 ml TAE buffer by heating the suspension. For staining, ethidium bromide was added into the mixture. Gels were run at 120 V for analytical and at 100 V for preparative use (200 mA respectively). For preparative gels, DNA fragments were excised and extracted from the gel using GK2041 gel recovery according to the manufacturer's protocol.
DNA digestion
Enzyme digestion system
| Component | Volume（μL） |
|---|---|
| DDW | - |
| 10×Buffer | 5 |
| Plamsid DNA | 1μg |
| Restriction endonuclease | 1 |
| Restriction endonuclease | 1 |
| Total volume | 50 |
Digestion conditions: 37℃ water bath overnigh
DNA ligation
The Ezmax one-step rapid cloning kit was used to recombine the purified PCR product and the linearized and purified vector.
| Component | Volume（μL） |
|---|---|
| 5×Buffer for Ezmax One-Step Cloning | 4 |
| Linearization vector | 8 |
| Exogenous DNA fragment | 2 |
| Ezmax recombinase | 2 |
| ddH2O | 4 |
| Total volume | 20 |
DNA ligation
DH5ɑ is activated on LB plates and is divided into single colonies
Pick a single colony and inoculate it in 2-3 mL of LB liquid medium overnight.
Inoculate the seed solution in fresh LB medium at 37℃for 1.5-3 h at a ratio of 1%. When the OD600 reaches about 0.4-0.6, the culture is taken out and placed on ice for 15-30 min, then transferred to pre-cooled 50 mL. Centrifuge the tube at 5000 rpm for 5 min at 4℃, discard the supernatant, and wash the cells once with sterile water.
Add 5mL of pre-cooled 0.1M calcium chloride solution to each centrifuge tube, and place it on ice for 15-30min, then gently shake the centrifuge tube until the bacteria at the bottom of the centrifuge tube are scattered (the whole process should be kept low temperature, gently Mix)
Then centrifuge at 5000 rpm for 5 min at 4℃, discard the supernatant, and then add an appropriate amount of pre-cooled 0.1 M calcium chloride solution containing 10% glycerol. Place on ice for 5 min, then shake the bacteria gently, Follow each 90 μL of the tube was dispensed into a pre-cooled 1.5 mL EP tube and stored at -80℃ for later use.
DNA ligation
A tube of prepared E. coli competent cells was placed in an ice water bath.
add 10 ul of DNA ligation solution to 90 μL competent cell suspension, mix gently, and place in an ice water bath for 30 min.
Mix well, pulse for 90s in a 42-degree water bath, transfer quickly to an ice water bath, add 800ul LB medium this tube.Then incubate for 45min on a 37-degree shaker.
Colony PCR
Pick 5-10 monoclonal clones from the culture dish and incubate them in a 10 mL centrifuge tube until a certain concentration of concentrated solution is used for PCR.
add 10 ul of DNA ligation solution to 90 μL competent cell suspension, mix gently, and place in an ice water bath for 30 min.
Mix well, pulse for 90s in a 42-degree water bath, transfer quickly to an ice water bath, add 800ul LB medium this tube.Then incubate for 45min on a 37-degree shaker.
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