Protocol
This is our Modeling DesignCloning
PCR
Reaction system:
1.
\(H_2 O\ \ 2\mu L\)
\(10\mathrm{x}\ \ Taq \ \ buffer \ \ 5\mu\mathrm{L}\)
\(2.5mM \ \ dNTP \ \ 1\mu\mathrm{L}\)
\(R+F-Primer \ \ 10\mu\mathrm{L}\)
\(Template \ \ 10\mu\mathrm{L}\)
\(Taq \ \ 2.5\mu\mathrm{L}\)
\(Universal \ \ DNA \ \ polymerase \ \ TransGen\)
2.
\(H_2 O\ \ 20\mu\mathrm{L}\)
\(5\mathrm{x}\ \ Taq\ \ buffer\ \ 10\mu\mathrm{L}\)
\(2.5mM\ \ dNTP\ \ 5\mu\mathrm{L}\)
\(R+F-Primer\ \ 44\mu\mathrm{L}\)
\(Template\ \ 10\mu\mathrm{L}\)
\(Taq\ \ 1\mu\mathrm{L}\)
3.
\(primeSTAR\ \ from\ \ Takara\)
\(H_2 O\ \ 21\mu\mathrm{L}\)
\(2\mathrm{x}\ \ primeSTAR\ \ 25m\mathrm{L}\)
\(R+F-Primer\ \ 2\mu\mathrm{L}\)
\(Template\ \ 2\mu\mathrm{L}\)
Process:
98°C 2min
\( \begin{equation} \left. \begin{array}{lcl} {98°C\ 10s} \\ {56°C\ 15s} \\{72°C\ 30s} \end{array} \right \} Cycle\ 35 \end{equation} \)
72°C 5min
4°C ---
98°C 2min
\(\begin{equation}\left. \begin{array}{lcl} {98°C\ 10s} \\ {55°C\ 5s} \\{72°C\ 8s} \end{array} \right\}Cycle\ 35\end{equation}\)
72°C 5min
15°C ---
Fusion PCR：
- basic PCR
- using the PCR product of step 1 as template does PCR
- using the PCR product of step 2 as template does PCR，but first five cycles don’t add primer, after first five cycles, the sixth cycle adds primer and continue PCR.
The system of step 2:
\(H_2 O\ \ 21\mu\mathrm{L}\)
\(2\mathrm{x}\ \ primeSTAR\ \ 25m\mathrm{L}\)
\(R+F-Primer\ \ 2\mu\mathrm{L}\)
\(Template①\ \ 1\mu\mathrm{L}\)
\(Template②\ \ 1\mu\mathrm{L}\)
Electrophoresis---Gel Purification
Material:
Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain
Protocol:
We used gelstain to stain the DNA and imaged it in a Transilluminator.
We used the gel extraction kit to get the objective fragment.
We used the DNA fragment purification kit to get the objective fragment.
Digestion
| \(50\mu\mathrm{L} \ \ \mathrm{reaction \ \ system}\) | |||||
|---|---|---|---|---|---|
| Reagent | 10x \(\ \mathrm{H \ buffer}\) | \(Eco\mathrm{R}\ \mathrm{I}\) | \(Pat\ \mathrm{I}\) | \(\mathrm{Plasmid}\) | \(\mathrm{H_2 O}\) |
| Dosage | \(5\mu\mathrm{L}\) | \(1.5\mu\mathrm{L}\) | \(1.5\mu\mathrm{L}\) | \(15\mu\mathrm{L}\) | \(27\mu\mathrm{L}\) |
| \(10\mu\mathrm{L} \ \ \mathrm{reaction \ \ system}\) | |||||
| Reagent | 10x \(\ \mathrm{H \ buffer}\) | \(Eco\mathrm{R}\ \mathrm{I}\) | \(Pat\ \mathrm{I}\) | \(\mathrm{Plasmid}\) | \(\mathrm{H_2 O}\) |
| Dosage | \(1\mu\mathrm{L}\) | \(0.3\mu\mathrm{L}\) | \(0.3\mu\mathrm{L}\) | \(3\mu\mathrm{L}\) | \(5.4\mu\mathrm{L}\) |
Ligation
| Ligation reaction system | ||||
|---|---|---|---|---|
| Reagent | DNA | Plasmid | T4 buffer | T4 ligase |
| Dosage | \(7\mu\mathrm{L}\) | \(1\mu\mathrm{L}\) | \(1\mu\mathrm{L}\) | \(1\mu\mathrm{L}\) |
LR reaction
1. Entry linearization
β2-TOPO（plasmid concentration 117ng/μL） NotI 37°C enzyme digestion for the night
| 50μL Single enzyme system | |
|---|---|
| 10x BufferH | 5μL |
| DNA | 20μL |
| ddH2O | 12.5μL |
| Enzyme | 2.5μL |
| 0.1%BSA | 5μL |
| 0.1%Triton X-100 | 5μL |
2. LR system (\(4\mu\mathrm{L}\)):
100 ng/ul linear Entry: 0.5 ul
destination vector: 1 ul (pCambia1300-nluc / pCambia1300-cluceach one)
LR Clonase II enzyme mix: 1 ul
ddH2O: 0.5 ul
mix slightly，water base for 5 h at 25°C
transform, 4 ul, reactant transform 50 ul competent cells
Transformation
Material:
| LB liquid medium | |||
|---|---|---|---|
| Reagent | Tryptone | Yeast extract powder | NaCl |
| Dosage | 10 g/L | 5 g/L | 10 μL |
Protocol:
preparation of the competent cells
1μL ligation product + 50μL cells
Heatshock of Trans5α(42°C,45s)
Put on ice(2min)
Add 500μL LB media and incubate for 1h(37°C, 150rpm)
Centrifuge at 4000rpm for 1min and remove 400μL supernatant
Resuspend the pellets using the left supernatant
Spread plates(with Kan;CHL)
Incubate for 12~16h(37°C)
Protein Expression
- Inoculated 3 mL LB media including relevant antibiotics with the monoclonal colony of expression plasmid, incubate for 12~16h(37°C, 190rpm)
- Inoculated 100 mL TM expression media including relevant antibiotics with the 1mL bacteria liquid, incubate for 3h(37°C, 250rpm,OD600=0.6~0.8)
- Add IPTG into it until its final concentration is 1mmol/L, incubate for 4~6h(37°C, 250rpm)
- Centrifuge at 6000rpm for 10min and remove supernatant
- Gather sediment, cryopreserve at -20°C
Material:
TM expression medium:1000mL PH=7.4
| Reagent | tryptone | Yeast extract powder | NaCl | glucose | glycerol |
| Dosage | 1.2g | 2.4g | 1.0g | 1.0g | 0.6mL |
Autoclaving 115℃，20min
Detetion
SDS-PAGE
Materials
| Gel | Tris-HCl | Acr/Bis 30% | SDS 10% | ddH2O | TEMED | AP 10% |
|---|---|---|---|---|---|---|
| Stacking Gel(4%) | pH=6.8 500μL | 500 μL | 25 μL | 1350μL | 2.5μL | 12.5μL |
| Running Gel(12%) | pH=8.8 1250μL | 2000 μL | 50 μL | 1675μL | 2.5μL | 25 μL |
| Running Gel(18%) | pH=8.8 1250μL | 3000 μL | 50 μL | 675 μL | 2.5μL | 25 μL |
| Running Buffer | |||
|---|---|---|---|
| Reagent | Tris-HCl | Glycine | (w/v) SDS |
| Dosage | 25 mmol/L | 0.192 mol/L | 0.1% |
Protocol
The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.
After ensuring that the equipment is waterproof, the 12 % (or 18%) running gel is mixed and filled into the chamber. Pipetting about 1 ml of H2O on top of the running gel to seal the gel.
After polymerization, the remaining H2O is removed and the 12 % stacking gel is filled on top. Insert a comb to create sample pockets.
After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via the SDS gel.
After loading the generated pockets with the samples, the stacking gel is run at 100 V and then running gel at 120 V.
Western Blot
System
| PBST:1000mL(PH=7.4) | |||||
|---|---|---|---|---|---|
| Reagent | NaCl(137mM) | KCl(2.7mM) | Na2HPO4(10mM) | K2HPO4(2mM) | Tween-20 |
| Dosage | 8g | 0.2g | 1.44g | 0.24g | 0.5mL |
| Imprint buffer：2000mL (PH=8.3) Transfer Buffer | |||
|---|---|---|---|
| Reagent | Tris | Gly | Methanol |
| Dosage | 6.06g | 28.8g | 400mL |
Protocol
- Transfer （Prepare transfer Buffer just before glue leaking, and precool at -20℃）.
- Put the transfer Buffer and the black subface of transfer splint downword, and lay a sponge in it. Several filter paper(three pieces of filter paper), glue(except Stacking Gel).
- Activation PVDF membrane in advance with anhydrous ethanol, and put it on the membrane.
- Three layers of filter paper, sponge, Squeeze out of the bubbles, turn tight.
- the black subface electric rotary groove stick to each other, put in ice.
- 110V，120min.
- 5% skim milk powder (prepared by PBST), block for a night.
- Dilute Primary antibody at the proportion of 1:2000 with 3% skim milk powder(add 0.02% sodium azide ), incubate 1h at the room temperature.
- PBST elute, wash with shocking for 5min , three times.
- Dilute Secondary antibody at the proportion of 1:2000 with 3% skim milk powder, incubate 1h at the room temperature.
- PBST elute, wash with shocking for 5min, three times.
- Color development.
