Team:BNU-China/Protocol

Team:BNU-CHINA - 2016.igem.org

PROTOCOL

Cloning

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:
  1. basic PCR
  2. using the PCR product of step 1 as template does PCR
  3. 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

2. LR system (\(4\mu\mathrm{L}\)):

100 ng/ul linear Entry: 0.5 ul

Transformation

Material:

Protocol: