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:
\(H_2 O\ \ 21\mu\mathrm{L}\)
\(2\mathrm{x}\ \ primeSTAR\ \ 25Μl\)
\(R+F-Primer\ \ 2\mu\mathrm{L}\)
\(Template①\ \ 1\mu\mathrm{L}\)
\(Template②\ \ 1\mu\mathrm{L}\)
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 | μL |
DNA | 20μL |
ddH2O | 12.5μL |
Enzyme | 2.5μL |
0.1%BSA | 5μL |
0.1%Triton X-100 | 5μL |