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

Ligation

LR reaction

1. Entry linearization

β2-TOPO（plasmid concentration 117ng/μL） NotI 37°C enzyme digestion for the night

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:

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

Autoclaving 115℃，20min

Detetion

SDS-PAGE

Materials

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

Protocol

Transfer （Prepare transfer Buffer just before glue leaking, and precool at -20℃）.

1. 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).
2. Activation PVDF membrane in advance with anhydrous ethanol, and put it on the membrane.
3. Three layers of filter paper, sponge, Squeeze out of the bubbles, turn tight.
4. the black subface electric rotary groove stick to each other, put in ice.
5. 110V，120min.
6. 5% skim milk powder (prepared by PBST), block for a night.
7. 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.
8. PBST elute, wash with shocking for 5min , three times.
9. Dilute Secondary antibody at the proportion of 1:2000 with 3% skim milk powder, incubate 1h at the room temperature.
10. PBST elute, wash with shocking for 5min, three times.
11. Color development.

Ni-beads protein purification

Protocol

1. Cut tips. Add 30μL Ni 6 fast flow Beads into 1.5mL EP.
2. Add 1mL NPI-10 buffer, mixing wash, sedimentate at low speed and wash 3 times.
3. Centrifuge and absorb supernatant into buffer. 4℃ binding 3h，rotate and mix.
4. After binding, Put on ice(5min),Centrifuge at 2000rpm for 1min.
5. absorb 80μL supernatant as control and remove the other supernatant, add 1mL NPI-20 washing, upside and down to mix, still standing, Centrifuge at 2000rpm for 1min(4℃), wash 3~5 times.
6. Add 500μL NPI-250 into Beads, rotate and mix for 15min, gather supernatant, add 500μL NPI-250 , rotate and mix for 15min, gather supernatant again.