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:

\(H_2 O\ \ 21\mu\mathrm{L}\)

\(2\mathrm{x}\ \ primeSTAR\ \ 25m\mathrm{L}\)

\(R+F-Primer\ \ 2\mu\mathrm{L}\)

\(Template①\ \ 1\mu\mathrm{L}\)

\(Template②\ \ 1\mu\mathrm{L}\)

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

  1. Inoculated 3 mL LB media including relevant antibiotics with the monoclonal colony of expression plasmid, incubate for 12~16h(37°C, 190rpm)
  2. 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)
  3. Add IPTG into it until its final concentration is 1mmol/L, incubate for 4~6h(37°C, 250rpm)
  4. Centrifuge at 6000rpm for 10min and remove supernatant
  5. 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