Difference between revisions of "Team:BNU-China/Protocol"

Latest revision as of 02:16, 20 October 2016

Team:BNU-CHINA - 2016.igem.org

Protocol

Cloning

PCR

Reaction system:

Universal DNA polymerase TransGen
ddH₂O	10x Taq Buffer	2.5 mM dNTP	R+F-Primer	Template	Taq
21.5 μL	5 μL	1 μL	10 μL	10 μL	2.5 μL

TaKaRa Taq^TM
ddH₂O	5x Taq Buffer	2.5 mM dNTP	R+F-Primer	Template	Taq
20 μL	10 μL	5 μL	4 μL	10 μL	1 μL

primeSTAR from TaKaRa^TM
ddH₂O	2x primeSTAR	R+F-Primer	Template
21 μL	25 mL	2 μL	2 μL

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
use the PCR product of step 1 as template to do PCR
use the PCR product of step 2 as template to do PCR, but first five cycles don’t add primer, add primer at the sixth cycle and continue PCR.

The system of step 2:

H₂O 21μL

2x primeSTAR 25mL

R+F-Primer 2 μL

Template① 1μL

Template② 1μL

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 μL reaction system
Reagent	10x \(\ \mathrm{H \ buffer}\)	\(Eco\mathrm{R}\ \mathrm{I}\)	\(Pst\ \mathrm{I}\)	\(\mathrm{Plasmid}\)	\(\mathrm{H_2 O}\)
Dosage	5 μL	1.5 μL	1.5 μL	15 μL	27 μL

10 μL reaction system
Reagent	10x \(\ \mathrm{H \ buffer}\)	\(Eco\mathrm{R}\ \mathrm{I}\)	\(Pst\ \mathrm{I}\)	\(\mathrm{Plasmid}\)	\(\mathrm{H_2 O}\)
Dosage	1 μL	0.3 μL	0.3 μL	3 μL	5.4 μL

Ligation

Ligation reaction system
Reagent	DNA	Plasmid	T4 buffer	T4 ligase
Dosage	7 μL	1 μL	1 μL	1 μL

LR reaction

1. Entry linearization

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

50 μL Single enzyme system
10x BufferH	5 μL
DNA	20 μL
ddH₂O	12.5 μL
Enzyme	2.5 μL
0.1%BSA	5 μL
0.1%Triton X-100	5 μL

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

100 ng/μL linear Entry: 0.5 μL

destination vector: 1 μL (pCambia1300-nluc / pCambia1300-cluceach one)

LR Clonase II enzyme mix: 1 μL

ddH₂O: 0.5 μL

mix slightly, water base for 5h at 25°C

transform, 4 μL, reactant transform 50 μL 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

1 μL ligation product + 50 μL cells

Heatshock of Trans5α(42°C, 45s)

Put on ice(2min)

Add 500 μL LB media and incubate for 1h(37°C, 150rpm)

Centrifuge at 4000 rpm for 1min and remove 400 μL 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 1 mL bacteria liquid, incubate for 3h(37°C, 250rpm,OD₆₀₀=0.6~0.8)
Add IPTG into it until its final concentration is 1 mmol/L, incubate for 4~6h(37°C, 250rpm)
Centrifuge at 6000 rpm for 10min and remove supernatant
Gather sediment, cryopreserve at -20°C

Material:

TM expression medium:1000 mL pH=7.4

Reagent	tryptone	Yeast extract powder	NaCl	glucose	glycerol
Dosage	1.2 g	2.4 g	1.0 g	1.0 g	0.6 mL

Autoclaving 115℃, 20min

Detection

SDS-PAGE

Materials

Gel	Tris-HCl	Acr/Bis 30%	SDS 10%	ddH₂O	TEMED	AP 10%
Stacking Gel(4%)	pH=6.8 500 μL	500 μL	25 μL	1350 μL	2.5 μL	12.5 μL
Running Gel(12%)	pH=8.8 1250 μL	2000 μL	50 μL	1675 μL	2.5 μL	25 μL
Running Gel(18%)	pH=8.8 1250 μL	3000 μL	50 μL	675 μL	2.5 μL	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:1000 mL(pH=7.4)
Reagent	NaCl(137mM)	KCl(2.7mM)	Na₂HPO₄(10mM)	K₂HPO₄(2mM)	Tween-20
Dosage	8 g	0.2 g	1.44 g	0.24 g	0.5 mL

Imprint buffer：2000 mL (pH=8.3) Transfer Buffer
Reagent	Tris	Gly	Methanol
Dosage	6.06 g	28.8 g	400 mL

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.

Ni-beads protein purification

NPI-10 buffer(1L) pH=8.0 filtration sterilization
Reagent	NaH₂PO₄·H₂O	NaCl	imidazole
Dosage	6.9	17.54	0.68

NPI-20 buffer(1L) pH=8.0 filtration sterilization
Reagent	NaH₂PO₄·H₂O	NaCl	imidazole
Dosage(g)	6.9	17.54	1.36

NPI-250 buffer(1L) pH=8.0 filtration sterilization
Reagent	NaH₂PO₄·H₂O	NaCl	imidazole
Dosage (g)	6.9	17.54	17.0

Protocol

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

Renaturation of the inclusion bodies

Binding buffer(1L) pH=8.0
Reagent	NaCl(500 mmol/L)	Na₃PO₄·12H₂O(20 mmol/L)	imidazole (20 mmol/L)
Dosage(g)	29.22	7.6	1.36

After cell disruption, sediment dissolves in binding buffer(8 mol/L urea)

Washing buffer(1 L)
Reagent	Tris-HCl(50 mmol/L)	EDTA(5 mmol/L)	NaCl(100 mmol/L)	Triton X-100(1%)
Dosage	100 mL	1.8612 g	5.844 g	10 mL

Induced expression
Collect sediment after ultrasonication, use washing buffer including 2, 3 mol/L urea to wash sediment in turn. Finally, use washing buffer including 8 mol/L urea to dissolve. Centrifuge and absorb supernatant, measure protein concentration and make it keep about 1 mg/mL. Dialyze these supernatant using binding buffer including concentration gradient urea(6,5,4,3,2,1,0.5 and 0 mol/L).

Extracting tubulin from porcine brains

Pick up 20 porcine brains from Beijing No.5 Meat Processing. For tubulin extraction experiment, the brains should be as fresh as possible. Take an ice box to store the brains. Avoid contact between the brains.
While getting the brains, another student should stay in the lab and prepared the centrifuge (set one at 4℃ and another at 37℃, also pre-warm the rotor). Put electronic balance, grinder, a graduated cylinder in refrigerator and pre-warm the glycerol and PEM in 37℃. prepare the fresh ATP and GTP buffer in the morning.
Clean the brain by tearing off the meninges and blood clots using kim wipes or by hand.
After cleaning, weigh the brains, put the brains in the blender, then add the same volume buffer PEM(with 1 mM DTT) in it accordingly.
Homogenate the brain for 3s, 10 times, time interval between two homogenate is 5s, in order to avoid destroy the tubulin because of high thermos.
Pour the homogenate into a flask, incubate in 4℃ for 30 min to depolymerize microtubules.
Pour the homogenate into tubes for Type 45Ti rotor and balance each tube.
Centrifuge at 8000 rpm for 40min at 4℃. Filter the supernatant with 4 gauzes. Then centrifuge the filtrate at 40000 g for 40min at 4℃.
Add 1/2 volume of warmed glycerol drop-wise with continuous shaking, mix gently but thoroughly. Add GTP (final concentration 0.1 mmol/L), MgCl₂ (final concentration 3 mmol/L), and EGTA (final concentration 1 mmol/L). Incubate in a 37℃ water bath for 1h, shake gently and occasionally.
Balance each tube and centrifuge at 100000 g for 40 min at 35℃.Discard supernatant, the pellet is crude extracts. Split charge them into 50 mL centrifuge tubes, each tube contains 5 g. Snap freeze the tubulin in 15 μL aliquots in liquid nitrogen and further stored at -80℃.
When going to do refined depuration, melt the freezing crude extract at 4℃ refrigerator on ice over night. Pop out 1 g of the pellet out of the tubes with a spatula. Put the pellets in the Dounce grinder, then add cold PEM in the tube to wash off residual pellets.
Re-suspend the pellets with grinder, keep the grinder on ice, and grinding occasionally. After 30min, pour out the solution and rinse the grinder with cold PEM. Total re-suspended volume is 5 mL.
Add GTP (final concentration 0.1 mmol/L). Place it on ice for 1h to depolymerize. Shake it occasionally.
Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4℃.
Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES( pH =6.9 ), DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl₂ (final concentration 1 mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.
Balance each tube and centrifuge at 100000 g for 1h at 35℃.
Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration 0.1 mmol/L). Place it on ice for 1h to depolymerize. Shake it occasionally.
Centrifuge the depolymerized tubulin at 100000 g for 40min at 4 ℃.
Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES ( pH= 6.9 ), DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl₂ (final concentration 1 mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.
Balance each tube and centrifuge at 100000 g for 1h at 35℃.
Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration 0.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it occasionally.
Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4℃.
Recover the supernatant, add equal volume polymerize buffer (containing 100 mmol/L PIPES-KOH, 2 mmol/L EGTA, 2 mmol/L MgCl₂, 2 mmol/L GTP and 60% glycerol).
Incubate in a 37℃ water bath for 1 h. Adding a series concentrations of taxol.
Balance each tube and centrifuge at 100000 g for 1 h at 35℃. The pellet is fine purified product.

Preparing STM samples

Use 200 # copper mesh to prepare the samples. This procedure was done in Institute of Biophysics, Chinese Academy of Science. Pretreat the copper mesh with Varian Plasma Cleaner PDC-32G.
Add protein solution on copper mesh. Keep still for 1 min.
Add 3 drops of uranyl acetate on parafilm. Mix the protein binding side with uranyl acetate thoroughly.
Store the sample carefully. Observe by transmission electron microscopy in Beijing Normal University.

OD₃₅₀ test

Take out the tubulin monomer solution after precision purification from -80℃, and melt on the ice.
Prepare 10 um tubulin monomer solution with the same concentration, and add isopyknic polymerization buffer (the formula is the same as the polymerization buffer used in precision purification). This step finished on the ice in order to avoid the influence of the Spontaneous tubulin polymerization because of the high room temperature.
Prepare a series concentration gradient of taxol solution and add the tubulin polymerization system.
Incubate for 1h at 37℃.
Use Nanodrop UV-IS to determinate absorbance value, and set measurement parameters 280 nm, 350 nm and 750 nm, but we only read at 350 nm.
Use isopyknic mixed solution of polymerization buffer and PEM buffer to calibrate baseline, each group of samples detect three times in parallel.

Improvement

Culture and collection

Use LB medium to preculture transformed media 5 ml for 12h, 200 rpm/ 37℃.
Culture 5 mL preculture media into 100 mL TB medium for about 3h until OD₆₀₀=0.4～0.6, 200 rpm/ 37℃.
Add arabinose or turn to 42℃ to induce P(3HB) expression for 72h, 220 rpm.
Collect cells and centrifuge for 3min, 5,000 rpm.
Remove supernatant and suspend with pure water.
Centrifuge again for 3min, 5,000 rpm and remove its supernatant.

Extract PHB¹

Centrifuge settings: 4000RPM, 10mins.
Scale as appropriate.
After each centrifuge step the supernatant should be poured off.

Resuspend precipitation in 10 mL Triton X-100(1% v/v in PBS) for 30mins at room temp.
Centrifuge, resuspend in 10 mL PBS.
Centrifuge, add 10 mL sodium hyperchlorite solution and incubate at 30˚C for 1 hour.
Centrifuge, wash with 10 mL 70% EtOH.
Allow powder to dry.

[1]: Shahryar Shakeri, Comparison of intracellular polyhydroxybutyrate granules formation between different bacterial cell subpopulations by flow cytometry, Jundishapur Journal of Microbiology 2011.

Agents formula

TB medium (1 L)
yeast extract	tryptone	K₂HPO₄	KH₂PO₄	Glycerol	Glucose	ddH₂O
24 g	12 g	72 mM	17 mM	8 mL	2%	add to 1 L

PBS (1 L)
NaCl	KCl	Na₂HPO₄·12H₂O	KH₂PO₄·3H₂O	pH	ddH₂O
8 g	0.2 g	3.63 g	0.31 g	adjust to 7.4	add to 1 L

Fluorescence Detection

Protein functional Test

Bacteria culturing and inducing for expression.
Collect supernatant after ultrasonication.

Concentrate the supernatant via ultra-filtering

Protein samples	α-Tubulin YNE	α-tubulin-YCE	β-tubulin	Aggregation buffer
Dosage	75 μL	75 μL	150 μL	300 μL

Protein samples	α-tubulin-YCE	β-tubulin- YNE	Aggregation buffer
Dosage	150 μL	150 μL	300 μL

Mixed the protein samples and add proper GTP, taxol to 200uM.
Use absolute recording spectrofluorometer with 514nm excitation.
Record light intensity in 520nm-530nm wavelength emission.

Taxol-concentration based assay

Bacteria culturing and inducing for expression
Collect supernatant after ultrasonication

Concentrate the supernatant via ultra-filtering

Protein samples	α-Tubulin YNE	α-tubulin-YCE	β-tubulin	Aggregation buffer
Dosage	75 μL	75 μL	150 μL	300 μL

Mixed the protein samples and add taxol respectively as the following table:

Taxol concentration (μM)

0

0.5

5

25

50

100

250

500

Use absolute recording spectrofluorometer with 514nm excitation
Record light intensity in 520nm-530nm wavelength emission.

@@ Line 1: / Line 1: @@
 {{BNU-CHINA/partials/header}}
 {{BNU-CHINA/partials/nav | TEAM=focus}}
+{{BNU-CHINA/article/theme | color=#660000}}
 <html>
-	<div class="main-container">
+    <div class="main-container">
-		<div id="page-heading" class="container-fluid page-heading"
+        <div id="page-heading" class="container-fluid page-heading"
-						 style="background-image: url(https://static.igem.org/mediawiki/2016/9/96/T--BNU-China--team.jpg);">
+                               style="background-image: url(https://static.igem.org/mediawiki/2016/a/a8/T--BNU-China--hand.jpg);">
-			<h3> PROTOCOL </h3>
+            <h3> Protocol </h3>
-		</div>
+        </div>
-		<div style="background-image: url(https://static.igem.org/mediawiki/2016/e/e5/T--BNU-China--landingImage.jpg); background-size: 100%;">
+        <div>
-			<div class="container page-story">
+            <div class="page-story">
-				<article id="modeling"
+                <article id="modeling"
-			 class="col-lg-10 col-lg-offset-1 col-md-12 col-md-offset-0 col-sm-offset-0 col-sm-12">
+                         class="col-lg-10 col-lg-offset-1 col-md-12 col-md-offset-0 col-sm-offset-0 col-sm-12">
-					<header class="page-header">
+                    <header class="page-header">
-						<h1>Protocol</h1>
+                        <h1>Protocol</h1>
-						<small id="secondary-page-header">This is our Protocol</small>
+                    </header>
-					</header>
+                    <h2>Cloning</h2>
-					<h2>Cloning</h2>
+                    <h3>PCR</h3>
-					<h3>PCR</h3>
+                    <h4>Reaction system:</h4>
-					<h4>Reaction system:</h4>
+                    <table class="table">
-					<table class="table">
+                        <tr>
-						<tr>
+                            <th style="text-align: center" colspan="6">Universal DNA polymerase TransGen</th>
-							<th style="text-align: center" colspan="6">Universal DNA polymerase TransGen</th>
+                        </tr>
-						</tr>
+                        <tr>
-						<tr>
+                            <td align="center">ddH<sub>2</sub>O</td>
-							<td align="center">ddH<sub>2</sub>O</td>
+                            <td align="center">10x Taq Buffer</td>
-							<td align="center">10x Taq Buffer</td>
+                            <td align="center">2.5 mM dNTP</td>
-							<td align="center">2.5mM dNTP</td>
+                            <td align="center">R+F-Primer</td>
-							<td align="center">R+F-Primer</td>
+                            <td align="center">Template</td>
-							<td align="center">Template</td>
+                            <td align="center">Taq</td>
-							<td align="center">Taq</td>
+                        </tr>
-						</tr>
+                        <tr>
-						<tr>
+                            <td align="center">21.5 μL</td>
-							<td align="center">2μL</td>
+                            <td align="center">5 μL</td>
-							<td align="center">5μL</td>
+                            <td align="center">1 μL</td>
-							<td align="center">1μL</td>
+                            <td align="center">10 μL</td>
-							<td align="center">10μL</td>
+                            <td align="center">10 μL</td>
-							<td align="center">10μL</td>
+                            <td align="center">2.5 μL</td>
-							<td align="center">2.5μL</td>
+                        </tr>
-						</tr>
+                    </table>
-					</table>
+                    <table class="table">
-					<table class="table">
+                        <tr>
-						<tr>
+                            <th style="text-align: center" colspan="6"><em>TaKaRa Taq</em><sup>TM</sup></th>
-							<th style="text-align: center" colspan="6"><em>TaKaRa Taq</em><sup>TM</sup></th>
+                        </tr>
-						</tr>
+                        <tr>
-						<tr>
+                            <td align="center">ddH<sub>2</sub>O</td>
-							<td align="center">ddH<sub>2</sub>O</td>
+                            <td align="center">5x Taq Buffer</td>
-							<td align="center">5x Taq Buffer</td>
+                            <td align="center">2.5 mM dNTP</td>
-							<td align="center">2.5mM dNTP</td>
+                            <td align="center">R+F-Primer</td>
-							<td align="center">R+F-Primer</td>
+                            <td align="center">Template</td>
-							<td align="center">Template</td>
+                            <td align="center">Taq</td>
-							<td align="center">Taq</td>
+                        </tr>
-						</tr>
+                        <tr>
-						<tr>
+                            <td align="center">20 μL</td>
-							<td align="center">20μL</td>
+                            <td align="center">10 μL</td>
-							<td align="center">10μL</td>
+                            <td align="center">5 μL</td>
-							<td align="center">5μL</td>
+                            <td align="center">4 μL</td>
-							<td align="center">44μL</td>
+                            <td align="center">10 μL</td>
-							<td align="center">10μL</td>
+                            <td align="center">1 μL</td>
-							<td align="center">1μL</td>
+                        </tr>
-						</tr>
+                    </table>
-					</table>
+                    <table class="table">
-					<table class="table">
+                        <tr>
-						<tr>
+                            <th style="text-align: center" colspan="4">primeSTAR from <em>TaKaRa</em><sup>TM</sup></th>
-							<th style="text-align: center" colspan="4">primeSTAR from <em>TaKaRa</em><sup>TM</sup></th>
+                        </tr>
-						</tr>
+                        <tr>
-						<tr>
+                            <td align="center">ddH<sub>2</sub>O</td>
-							<td align="center">ddH<sub>2</sub>O</td>
+                            <td align="center">2x primeSTAR</td>
-							<td align="center">2x primeSTAR</td>
+                            <td align="center">R+F-Primer</td>
-							<td align="center">R+F-Primer</td>
+                            <td align="center">Template</td>
-							<td align="center">Template</td>
+                        </tr>
-						</tr>
+                        <tr>
-						<tr>
+                            <td align="center">21 μL</td>
-							<td align="center">21μL</td>
+                            <td align="center">25 mL</td>
-							<td align="center">25ML</td>
+                            <td align="center">2 μL</td>
-							<td align="center">2μL</td>
+                            <td align="center">2 μL</td>
-							<td align="center">2μL</td>
+                        </tr>
-						</tr>
+                    </table>
-					</table>
+                    <h4>Process:</h4>
-					<h4>Process:</h4>
+                    <p>98&#176;C 2min </p>
-					<p>98&#176;C 2min </p>
+                    <p> \( \begin{equation} \left. \begin{array}{lcl} {98&#176;C\ 10s} \\ {56&#176;C\ 15s} \\{72&#176;C\
-					<p> \( \begin{equation} \left. \begin{array}{lcl} {98&#176;C\ 10s} \\ {56&#176;C\ 15s} \\{72&#176;C\
+s} \end{array} \right \} Cycle\ 35 \end{equation} \) </p>
-s} \end{array} \right \} Cycle\ 35 \end{equation} \) </p>
+                    <p>72&#176;C 5min </p>
-					<p>72&#176;C 5min </p>
+                    <p>4&#176;C --- </p>
-					<p>4&#176;C --- </p>
+                    <p>98&#176;C 2min </p>
-					<p>98&#176;C 2min </p>
+                    <p>\(\begin{equation}\left. \begin{array}{lcl} {98&#176;C\ 10s} \\ {55&#176;C\ 5s} \\{72&#176;C\ 8s}
-					<p>\(\begin{equation}\left. \begin{array}{lcl} {98&#176;C\ 10s} \\ {55&#176;C\ 5s} \\{72&#176;C\ 8s}
+                    \end{array} \right\}Cycle\ 35\end{equation}\)</p>
-					\end{array} \right\}Cycle\ 35\end{equation}\)</p>
+                    <p>72&#176;C 5min </p>
-					<p>72&#176;C 5min </p>
+                    <p>15&#176;C --- </p>
-					<p>15&#176;C --- </p>
+                    <h4>Fusion PCR：</h4>
-					<h5>Fusion PCR：</h5>
+                    <ol>
-					<ol>
+                        <li> basic PCR</li>
-						<li> basic PCR</li>
+                        <li> use the PCR product of step 1 as template to do PCR</li>
-						<li> using the PCR product of step 1 as template does PCR</li>
+                        <li> use the PCR product of step 2 as template to do PCR, but first five cycles don’t add primer,
-						<li> using the PCR product of step 2 as template does PCR，but first five cycles don’t add primer,
+                            add primer at the sixth cycle and continue PCR.
-							after first five cycles, the sixth cycle adds primer and continue PCR.
+                        </li>
-						</li>
+                    </ol>
-					</ol>
+                    <h5> The system of step 2: </h5>
-					<h5> The system of step 2: </h5>
+                    <p>H<sub>2</sub>O 21μL</p>
-					<p>\(H_2 O\ \ 21\mu\mathrm{L}\)</p>
+                    <p>2x primeSTAR 25mL</p>
-					<p>\(2\mathrm{x}\ \ primeSTAR\ \ 25m\mathrm{L}\)</p>
+                    <p>R+F-Primer 2 μL</p>
-					<p>\(R+F-Primer\ \ 2\mu\mathrm{L}\)</p>
+                    <p>Template① 1μL</p>
-					<p>\(Template①\ \ 1\mu\mathrm{L}\)</p>
+                    <p>Template② 1μL</p>
-					<p>\(Template②\ \ 1\mu\mathrm{L}\)</p>
+                    <h3>Electrophoresis---Gel Purification</h3>
+                    <h4>Material:</h4>
+                    <p>Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain</p>
+                    <h4>Protocol:</h4>
+                    <p>We used gelstain to stain the DNA and imaged it in a Transilluminator.</p>
+                    <p>We used the gel extraction kit to get the objective fragment.</p>
+                    <p>We used the DNA fragment purification kit to get the objective fragment.</p>
+                    <h3>Digestion</h3>
+                    <table class="table">
+                        <tr>
+                            <th colspan="6" style="text-align: center">50 μL reaction system</th>
+                        </tr>
+                        <tr>
+                            <td align="center">Reagent</td>
+                            <td align="center">10x \(\ \mathrm{H \ buffer}\)</td>
+                            <td align="center">\(Eco\mathrm{R}\ \mathrm{I}\)</td>
+                            <td align="center">\(Pst\ \mathrm{I}\)</td>
+                            <td align="center">\(\mathrm{Plasmid}\)</td>
+                            <td align="center">\(\mathrm{H_2 O}\)</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage</td>
+                            <td align="center">5 μL</td>
+                            <td align="center">1.5 μL</td>
+                            <td align="center">1.5 μL</td>
+                            <td align="center">15 μL</td>
+                            <td align="center">27 μL</td>
+                        </tr>
+                    </table>
+                    <table class="table">
+                        <tr>
+                            <th colspan="6" style="text-align: center">10 μL reaction system</th>
+                        </tr>
+                        <tr>
+                            <td align="center">Reagent</td>
+                            <td align="center">10x \(\ \mathrm{H \ buffer}\)</td>
+                            <td align="center">\(Eco\mathrm{R}\ \mathrm{I}\)</td>
+                            <td align="center">\(Pst\ \mathrm{I}\)</td>
+                            <td align="center">\(\mathrm{Plasmid}\)</td>
+                            <td align="center">\(\mathrm{H_2 O}\)</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage</td>
+                            <td align="center">1 μL</td>
+                            <td align="center">0.3 μL</td>
+                            <td align="center">0.3 μL</td>
+                            <td align="center">3 μL</td>
+                            <td align="center">5.4 μL</td>
+                        </tr>
+                    </table>
+                    <h2>Ligation</h2>
+                    <table class="table">
+                        <tr>
+                            <th colspan="5" style="text-align: center">Ligation reaction system</th>
+                        </tr>
+                        <tr>
+                            <td align="center">Reagent</td>
+                            <td align="center">DNA</td>
+                            <td align="center">Plasmid</td>
+                            <td align="center">T4 buffer</td>
+                            <td align="center">T4 ligase</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage</td>
+                            <td align="center">7 μL</td>
+                            <td align="center">1 μL</td>
+                            <td align="center">1 μL</td>
+                            <td align="center">1 μL</td>
+                        </tr>
+                    </table>
+                    <h3>LR reaction</h3>
+                    <h4>1. Entry linearization</h4>
+                    <p>β2-TOPO (plasmid concentration 117 ng/μL)NotI 37&#176;C enzyme digestion for the night</p>
+                    <table class="table">
+                        <tr>
+                            <th style="text-align: center" colspan="2"> 50 μL Single enzyme system</th>
+                        </tr>
+                        <tr>
+                            <td align="center">10x BufferH</td>
+                            <td align="center">5 μL</td>
+                        </tr>
+                        <tr>
+                            <td align="center">DNA</td>
+                            <td align="center">20 μL</td>
+                        </tr>
+                        <tr>
+                            <td align="center">ddH<sub>2</sub>O</td>
+                            <td align="center">12.5 μL</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Enzyme</td>
+                            <td align="center">2.5 μL</td>
+                        </tr>
+                        <tr>
+                            <td align="center">0.1%BSA</td>
+                            <td align="center">5 μL</td>
+                        </tr>
+                        <tr>
+                            <td align="center">0.1%Triton X-100</td>
+                            <td align="center">5 μL</td>
+                        </tr>
+                    </table>
-					<h3>Electrophoresis---Gel Purification</h3>
+                    <h4>2. LR system (\(4\mu\mathrm{L}\)):</h4>
-					<h4>Material:</h4>
+                    <p>100 ng/μL linear Entry: 0.5 μL</p>
-					<p>Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain</p>
+                    <p>destination vector: 1 μL (pCambia1300-nluc / pCambia1300-cluceach one)</p>
-					<h4>Protocol:</h4>
+                    <p>LR Clonase II enzyme mix: 1 μL</p>
-					<p>We used gelstain to stain the DNA and imaged it in a Transilluminator.</p>
+                    <p>ddH<sub>2</sub>O: 0.5 μL</p>
-					<p>We used the gel extraction kit to get the objective fragment.</p>
+                    <p>mix slightly, water base for 5h at 25&#176;C </p>
-					<p>We used the DNA fragment purification kit to get the objective fragment.</p>
+                    <p>transform, 4 μL, reactant transform 50 μL competent cells</p>
-					<h3>Digestion</h3>
-					<table class="table">
-						<tr>
-							<th colspan="6" style="text-align: center">50μL reaction system</th>
-						</tr>
-						<tr>
-							<td align="center">Reagent</td>
-							<td align="center">10x \(\ \mathrm{H \ buffer}\)</td>
-							<td align="center">\(Eco\mathrm{R}\ \mathrm{I}\)</td>
-							<td align="center">\(Pat\ \mathrm{I}\)</td>
-							<td align="center">\(\mathrm{Plasmid}\)</td>
-							<td align="center">\(\mathrm{H_2 O}\)</td>
-						</tr>
-						<tr>
-							<td align="center">Dosage</td>
-							<td align="center">5μL</td>
-							<td align="center">1.5μL</td>
-							<td align="center">1.5μL</td>
-							<td align="center">15μL</td>
-							<td align="center">27μL</td>
-						</tr>
-					</table>
-					<table class="table">
-						<tr>
-							<th colspan="6" style="text-align: center">10μL  reaction  system</th>
-						</tr>
-						<tr>
-							<td align="center">Reagent</td>
-							<td align="center">10x \(\ \mathrm{H \ buffer}\)</td>
-							<td align="center">\(Eco\mathrm{R}\ \mathrm{I}\)</td>
-							<td align="center">\(Pat\ \mathrm{I}\)</td>
-							<td align="center">\(\mathrm{Plasmid}\)</td>
-							<td align="center">\(\mathrm{H_2 O}\)</td>
-						</tr>
-						<tr>
-							<td align="center">Dosage</td>
-							<td align="center">1μL</td>
-							<td align="center">0.3μL</td>
-							<td align="center">0.3μL</td>
-							<td align="center">3μL</td>
-							<td align="center">5.4μL</td>
-						</tr>
-					</table>
-					<h2>Ligation</h2>
+                    <h2>Transformation</h2>
-					<table class="table">
-						<tr>
-							<th colspan="5" style="text-align: center">Ligation reaction system</th>
-						</tr>
-						<tr>
-							<td align="center">Reagent</td>
-							<td align="center">DNA</td>
-							<td align="center">Plasmid</td>
-							<td align="center">T4 buffer</td>
-							<td align="center">T4 ligase</td>
-						</tr>
-						<tr>
-							<td align="center">Dosage</td>
-							<td align="center">7μL</td>
-							<td align="center">1μL</td>
-							<td align="center">1μL</td>
-							<td align="center">1μL</td>
-						</tr>
-					</table>
-					<h3>LR reaction</h3>
+                    <h3>Material:</h3>
+                    <table class="table">
+                        <tr>
+                            <th colspan="4" style="text-align: center">LB liquid medium</th>
+                        </tr>
+                        <tr>
+                            <td align="center">Reagent</td>
+                            <td align="center">Tryptone</td>
+                            <td align="center">Yeast extract powder</td>
+                            <td align="center">NaCl</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage</td>
+                            <td align="center">10 g/L</td>
+                            <td align="center">5 g/L</td>
+                            <td align="center">10 μL</td>
+                        </tr>
+                    </table>
-					<h4>1. Entry linearization</h4>
+                    <h3>Protocol:</h3>
-					<p>β2-TOPO（plasmid concentration 117ng/μL） NotI 37&#176;C enzyme digestion for the night</p>
+                    <p> Preparation of the competent cells </p>
-					<table class="table">
+                    <p> 1 μL ligation product + 50 μL cells </p>
-						<tr>
+                    <p>Heatshock of Trans5α(42&#176;C, 45s)</p>
-							<th style="text-align: center" colspan="2"> 50μL Single enzyme system</th>
+                    <p>Put on ice(2min)</p>
-						</tr>
+                    <p>Add 500 μL LB media and incubate for 1h(37&#176;C, 150rpm)</p>
-						<tr>
+                    <p>Centrifuge at 4000 rpm for 1min and remove 400 μL supernatant</p>
-							<td align="center">10x BufferH</td>
+                    <p>Resuspend the pellets using the left supernatant</p>
-							<td align="center">5μL</td>
+                    <p>Spread plates(with Kan;Chl)</p>
-						</tr>
+                    <p>Incubate for 12~16h(37&#176;C)</p>
-						<tr>
-							<td align="center">DNA</td>
-							<td align="center">20μL</td>
-						</tr>
-						<tr>
-							<td align="center">ddH<sub>2</sub>O</td>
-							<td align="center">12.5μL</td>
-						</tr>
-						<tr>
-							<td align="center">Enzyme</td>
-							<td align="center">2.5μL</td>
-						</tr>
-						<tr>
-							<td align="center">0.1%BSA</td>
-							<td align="center">5μL</td>
-						</tr>
-						<tr>
-							<td align="center">0.1%Triton X-100</td>
-							<td align="center">5μL</td>
-						</tr>
-					</table>
-					<h4>2. LR system (\(4\mu\mathrm{L}\)):</h4>
+                    <h2>Protein expression</h2>
-					<p>100 ng/ul linear Entry: 0.5 ul</p>
+                    <ol>
-					<p>destination vector: 1 ul (pCambia1300-nluc / pCambia1300-cluceach one)</p>
+                        <li>Inoculated 3 mL LB media including relevant antibiotics with the monoclonal colony of expression
-					<p>LR Clonase II enzyme mix: 1 ul</p>
+                            plasmid, incubate for 12~16h(37&#176;C, 190rpm)
-					<p>ddH2O: 0.5 ul</p>
+                        </li>
-					<p>mix slightly，water base for 5 h at 25&#176;C </p>
+                        <li>Inoculated 100 mL TM expression media including relevant antibiotics with the 1 mL bacteria
-					<p>transform, 4 ul, reactant transform 50 ul competent cells</p>
+                            liquid, incubate for 3h(37&#176;C, 250rpm,OD<sub>600</sub>=0.6~0.8)
+                        </li>
+                        <li>Add IPTG into it until its final concentration is 1 mmol/L, incubate for 4~6h(37&#176;C,
+rpm)
+                        </li>
+                        <li>Centrifuge at 6000 rpm for 10min and remove supernatant</li>
+                        <li>Gather sediment, cryopreserve at -20&#176;C</li>
+                    </ol>
+                    <p>Material:</p>
+                    <p>TM expression medium:1000 mL pH=7.4</p>
-					<h2>Transformation</h2>
+                    <table class="table">
+                        <tr>
+                            <td align="center">Reagent</td>
+                            <td align="center">tryptone</td>
+                            <td align="center">Yeast extract powder</td>
+                            <td align="center">NaCl</td>
+                            <td align="center">glucose</td>
+                            <td align="center">glycerol</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage</td>
+                            <td align="center">1.2 g</td>
+                            <td align="center">2.4 g</td>
+                            <td align="center">1.0 g</td>
+                            <td align="center">1.0 g</td>
+                            <td align="center">0.6 mL</td>
+                        </tr>
+                    </table>
+                    <p>Autoclaving 115℃, 20min</p>
-					<h3>Material:</h3>
+                    <h2>Detection</h2>
-					<table class="table">
+                    <h3>SDS-PAGE</h3>
-						<tr>
+                    <h4>Materials</h4>
-							<th colspan="4" style="text-align: center">LB liquid medium</th>
+                    <table class="table">
-						</tr>
+                        <tr>
-						<tr>
+                            <th style="text-align: center">Gel</th>
-							<td align="center">Reagent</td>
+                            <th style="text-align: center">Tris-HCl</th>
-							<td align="center">Tryptone</td>
+                            <th style="text-align: center">Acr/Bis 30%</th>
-							<td align="center">Yeast extract powder</td>
+                            <th style="text-align: center">SDS 10%</th>
-							<td align="center">NaCl</td>
+                            <th style="text-align: center">ddH<sub>2</sub>O</th>
-						</tr>
+                            <th style="text-align: center">TEMED</th>
-						<tr>
+                            <th style="text-align: center">AP 10%</th>
-							<td align="center">Dosage</td>
+                        </tr>
-							<td align="center">10 g/L</td>
+                        <tr>
-							<td align="center">5 g/L</td>
+                            <td align="center">Stacking Gel(4%)</td>
-							<td align="center">10 μL</td>
+                            <td align="center">pH=6.8 500 μL</td>
-						</tr>
+                            <td align="center">500 μL</td>
-					</table>
+                            <td align="center">25 μL</td>
+                            <td align="center">1350 μL</td>
+                            <td align="center">2.5 μL</td>
+                            <td align="center">12.5 μL</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Running Gel(12%)</td>
+                            <td align="center">pH=8.8 1250 μL</td>
+                            <td align="center">2000 μL</td>
+                            <td align="center">50 μL</td>
+                            <td align="center">1675 μL</td>
+                            <td align="center">2.5 μL</td>
+                            <td align="center">25 μL</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Running Gel(18%)</td>
+                            <td align="center">pH=8.8 1250 μL</td>
+                            <td align="center">3000 μL</td>
+                            <td align="center">50 μL</td>
+                            <td align="center">675 μL</td>
+                            <td align="center">2.5 μL</td>
+                            <td align="center">25 μL</td>
+                        </tr>
+                    </table>
+                    <table class="table">
+                        <tr>
+                            <th colspan="4" style="text-align: center">Running Buffer</th>
+                        </tr>
+                        <tr>
+                            <td align="center">Reagent</td>
+                            <td align="center">Tris-HCl</td>
+                            <td align="center">Glycine</td>
+                            <td align="center">(w/v) SDS</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage</td>
+                            <td align="center">25 mmol/L</td>
+                            <td align="center">0.192 mol/L</td>
+                            <td align="center">0.1%</td>
+                        </tr>
+                    </table>
+                    <h4>Protocol</h4>
+                    <p>The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.</p>
+                    <p>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 H<sub>2</sub>O on top of the running gel to seal the gel.
+                    </p>
+                    <p>After polymerization, the remaining H<sub>2</sub>O is removed and the 12% stacking gel is filled on
+                    top. Insert
+                    a comb to create sample pockets.</p>
+                    <p>After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via
+                    the SDS gel. </p>
+                    <p>After loading the generated pockets with the samples, the stacking gel is run at 100 V and then
+                    running gel at 120 V. </p>
-					<h3>Protocol:</h3>
+                    <h3>Western Blot</h3>
-					<p> preparation of the competent cells </p>
+                    <h4>System</h4>
-					<p> 1μL ligation product + 50μL cells </p>
+                    <table class="table">
-					<p>Heatshock of Trans5α(42&#176;C,45s)</p>
+                        <tr>
-					<p>Put on ice(2min)</p>
+                            <th colspan="6" style="text-align: center"> PBST:1000 mL(pH=7.4)</th>
-					<p>Add 500μL LB media and incubate for 1h(37&#176;C, 150rpm)</p>
+                        </tr>
-					<p>Centrifuge at 4000rpm for 1min and remove 400μL supernatant</p>
+                        <tr>
-					<p>Resuspend the pellets using the left supernatant</p>
+                            <td align="center">Reagent</td>
-					<p>Spread plates(with Kan;CHL)</p>
+                            <td align="center">NaCl(137mM)</td>
-					<p>Incubate for 12~16h(37&#176;C)</p>
+                            <td align="center">KCl(2.7mM)</td>
+                            <td align="center">Na<sub>2</sub>HPO<sub>4</sub>(10mM)</td>
+                            <td align="center">K<sub>2</sub>HPO<sub>4</sub>(2mM)</td>
+                            <td align="center">Tween-20</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage</td>
+                            <td align="center">8 g</td>
+                            <td align="center">0.2 g</td>
+                            <td align="center">1.44 g</td>
+                            <td align="center">0.24 g</td>
+                            <td align="center">0.5 mL</td>
+                        </tr>
-					<h2>Protein Expression</h2>
+                    </table>
-					<ol>
-						<li>Inoculated 3 mL LB media including relevant antibiotics with the monoclonal colony of expression
-							plasmid, incubate for 12~16h(37&#176;C, 190rpm)
-						</li>
-						<li>Inoculated 100 mL TM expression media including relevant antibiotics with the 1mL bacteria
-							liquid, incubate for 3h(37&#176;C, 250rpm,OD600=0.6~0.8)
-						</li>
-						<li>Add IPTG into it until its final concentration is 1mmol/L, incubate for 4~6h(37&#176;C,
-rpm)
-						</li>
-						<li>Centrifuge at 6000rpm for 10min and remove supernatant</li>
-						<li>Gather sediment, cryopreserve at -20&#176;C</li>
-					</ol>
-					<p>Material:</p>
-					<p>TM expression medium:1000mL PH=7.4</p>
-					<table class="table">
+                    <table class="table">
-						<tr>
+                        <tr>
-							<td align="center">Reagent</td>
+                            <th colspan="4" style="text-align: center">Imprint buffer：2000 mL (pH=8.3) Transfer Buffer</th>
-							<td align="center">tryptone</td>
+                        </tr>
-							<td align="center">Yeast extract powder</td>
+                        <tr>
-							<td align="center">NaCl</td>
+                            <td align="center">Reagent</td>
-							<td align="center">glucose</td>
+                            <td align="center">Tris</td>
-							<td align="center">glycerol</td>
+                            <td align="center">Gly</td>
-						</tr>
+                            <td align="center">Methanol</td>
-						<tr>
+                        </tr>
-							<td align="center">Dosage</td>
+                        <tr>
-							<td align="center">1.2g</td>
+                            <td align="center">Dosage</td>
-							<td align="center">2.4g</td>
+                            <td align="center">6.06 g</td>
-							<td align="center">1.0g</td>
+                            <td align="center">28.8 g</td>
-							<td align="center">1.0g</td>
+                            <td align="center">400 mL</td>
-							<td align="center">0.6mL</td>
+                        </tr>
-						</tr>
+                    </table>
-					</table>
-					<p>Autoclaving 115℃，20min</p>
-					<h2>Detection</h2>
+                    <h4>Protocol</h4>
-					<h3>SDS-PAGE</h3>
+                    <p>Transfer (Prepare transfer Buffer just before glue leaking, and precool at -20℃).</p>
-					<h4>Materials</h4>
+                    <ol>
-					<table class="table">
+                        <li>Put the transfer Buffer and the black subface of transfer splint downword, and lay a sponge in
-						<tr>
+                            it. Several filter paper(three pieces of filter paper), glue(except Stacking Gel).
-							<th style="text-align: center">Gel</th>
+                        </li>
-							<th style="text-align: center">Tris-HCl</th>
+                        <li>Activation PVDF membrane in advance with anhydrous ethanol, and put it on the membrane.</li>
-							<th style="text-align: center">Acr/Bis 30%</th>
+                        <li>Three layers of filter paper, sponge, Squeeze out of the bubbles, turn tight.</li>
-							<th style="text-align: center">SDS 10%</th>
+                        <li>The black subface electric rotary groove stick to each other, put in ice.</li>
-							<th style="text-align: center">ddH<sub>2</sub>O</th>
+                        <li>110V, 120min.</li>
-							<th style="text-align: center">TEMED</th>
+                        <li>5% skim milk powder (prepared by PBST), block for a night.</li>
-							<th style="text-align: center">AP 10%</th>
+                        <li>Dilute Primary antibody at the proportion of 1:2000 with 3% skim milk powder(add 0.02% sodium
-						</tr>
+                            azide ), incubate 1h at the room temperature.
-						<tr>
+                        </li>
-							<td align="center">Stacking Gel(4%)</td>
+                        <li>PBST elute, wash with shocking for 5min , three times.</li>
-							<td align="center">pH=6.8 500μL</td>
+                        <li>Dilute Secondary antibody at the proportion of 1:2000 with 3% skim milk powder, incubate 1h at
-							<td align="center">500 μL</td>
+                            the room temperature.
-							<td align="center">25 μL</td>
+                        </li>
-							<td align="center">1350μL</td>
+                        <li>PBST elute, wash with shocking for 5min, three times.</li>
-							<td align="center">2.5μL</td>
+                        <li>Color development.</li>
-							<td align="center">12.5μL</td>
+                    </ol>
-						</tr>
-						<tr>
-							<td align="center">Running Gel(12%)</td>
-							<td align="center">pH=8.8 1250μL</td>
-							<td align="center">2000 μL</td>
-							<td align="center">50 μL</td>
-							<td align="center">1675μL</td>
-							<td align="center">2.5μL</td>
-							<td align="center">25 μL</td>
-						</tr>
-						<tr>
-							<td align="center">Running Gel(18%)</td>
-							<td align="center">pH=8.8 1250μL</td>
-							<td align="center">3000 μL</td>
-							<td align="center">50 μL</td>
-							<td align="center">675 μL</td>
-							<td align="center">2.5μL</td>
-							<td align="center">25 μL</td>
-						</tr>
-					</table>
-					<table class="table">
-						<tr>
-							<th colspan="4" style="text-align: center">Running Buffer</th>
-						</tr>
-						<tr>
-							<td align="center">Reagent</td>
-							<td align="center">Tris-HCl</td>
-							<td align="center">Glycine</td>
-							<td align="center">(w/v) SDS</td>
-						</tr>
-						<tr>
-							<td align="center">Dosage</td>
-							<td align="center">25 mmol/L</td>
-							<td align="center">0.192 mol/L</td>
-							<td align="center">0.1%</td>
-						</tr>
-					</table>
-					<h4>Protocol</h4>
-					<p>The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.</p>
-					<p>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. </p>
-					<p>After polymerization, the remaining H<sub>2</sub>O is removed and the 12 % stacking gel is filled on top. Insert
-					a comb to create sample pockets.</p>
-					<p>After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via
-					the SDS gel. </p>
-					<p>After loading the generated pockets with the samples, the stacking gel is run at 100 V and then
-					running gel at 120 V. </p>
-					<h3>Western Blot</h3>
+                    <h2>Ni-beads protein purification</h2>
-					<h4>System</h4>
-					<table class="table">
-						<tr>
-							<th colspan="6" style="text-align: center"> PBST:1000mL(PH=7.4)</th>
-						</tr>
-						<tr>
-							<td align="center">Reagent</td>
-							<td align="center">NaCl(137mM)</td>
-							<td align="center">KCl(2.7mM)</td>
-							<td align="center">Na<sub>2</sub>HPO<sub>4</sub>(10mM)</td>
-							<td align="center">K<sub>2</sub>HPO<sub>4</sub>(2mM)</td>
-							<td align="center">Tween-20</td>
-						</tr>
-						<tr>
-							<td align="center">Dosage</td>
-							<td align="center">8g</td>
-							<td align="center">0.2g</td>
-							<td align="center">1.44g</td>
-							<td align="center">0.24g</td>
-							<td align="center">0.5mL</td>
-						</tr>
-					</table>
-					<table class="table">
+                    <table class="table">
-						<tr>
+                        <tr>
-							<th colspan="4" style="text-align: center">Imprint buffer：2000mL (PH=8.3) Transfer Buffer</th>
+                            <th style="text-align: center" colspan="4"> NPI-10 buffer(1L) pH=8.0 filtration sterilization
-						</tr>
+                            </th>
-						<tr>
+                        </tr>
-							<td align="center">Reagent</td>
+                        <tr>
-							<td align="center">Tris</td>
+                            <td align="center">Reagent</td>
-							<td align="center">Gly</td>
+                            <td align="center">NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O</td>
-							<td align="center">Methanol</td>
+                            <td align="center">NaCl</td>
-						</tr>
+                            <td align="center">imidazole</td>
-						<tr>
+                        </tr>
-							<td align="center">Dosage</td>
+                        <tr>
-							<td align="center">6.06g</td>
+                            <td align="center">Dosage</td>
-							<td align="center">28.8g</td>
+                            <td align="center">6.9</td>
-							<td align="center">400mL</td>
+                            <td align="center">17.54</td>
-						</tr>
+                            <td align="center">0.68</td>
-					</table>
+                        </tr>
+                    </table>
-					<h4>Protocol</h4>
+                    <table class="table">
-					<p>Transfer （Prepare transfer Buffer just before glue leaking, and precool at -20℃）.</p>
+                        <tr>
-					<ol>
+                            <th style="text-align: center" colspan="4">NPI-20 buffer(1L) pH=8.0 filtration sterilization</th>
-						<li>Put the transfer Buffer and the black subface of transfer splint downword, and lay a sponge in
+                        </tr>
-							it. Several filter paper(three pieces of filter paper), glue(except Stacking Gel).
+                        <tr>
-						</li>
+                            <td align="center">Reagent</td>
-						<li>Activation PVDF membrane in advance with anhydrous ethanol, and put it on the membrane.</li>
+                            <td align="center">NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O</td>
-						<li>Three layers of filter paper, sponge, Squeeze out of the bubbles, turn tight.</li>
+                            <td align="center">NaCl</td>
-						<li>the black subface electric rotary groove stick to each other, put in ice.</li>
+                            <td align="center">imidazole</td>
-						<li>110V，120min.</li>
+                        </tr>
-						<li>5% skim milk powder (prepared by PBST), block for a night.</li>
+                        <tr>
-						<li>Dilute Primary antibody at the proportion of 1:2000 with 3% skim milk powder(add 0.02% sodium
+                            <td align="center">Dosage(g)</td>
-							azide ), incubate 1h at the room temperature.
+                            <td align="center">6.9</td>
-						</li>
+                            <td align="center">17.54</td>
-						<li>PBST elute, wash with shocking for 5min , three times.</li>
+                            <td align="center">1.36</td>
-						<li>Dilute Secondary antibody at the proportion of 1:2000 with 3% skim milk powder, incubate 1h at
+                        </tr>
-							the room temperature.
+                    </table>
-						</li>
-						<li>PBST elute, wash with shocking for 5min, three times.</li>
-						<li>Color development.</li>
-					</ol>
-					<h2>Ni-beads protein purification</h2>
+                    <table class="table">
+                        <tr>
+                            <th style="text-align: center" colspan="4">NPI-250 buffer(1L) pH=8.0 filtration sterilization
+                            </th>
+                        </tr>
+                        <tr>
+                            <td align="center">Reagent</td>
+                            <td align="center">NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O</td>
+                            <td align="center">NaCl</td>
+                            <td align="center">imidazole</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage (g)</td>
+                            <td align="center">6.9</td>
+                            <td align="center">17.54</td>
+                            <td align="center">17.0</td>
+                        </tr>
+                    </table>
+                    <h4>Protocol</h4>
+                    <ol>
+                        <li>Cut tips. Add 30 μL Ni 6 fast flow Beads into 1.5 mL EP.</li>
+                        <li>Add 1 mL NPI-10 buffer, mixing wash, sedimentate at low speed and wash 3 times.</li>
+                        <li>Centrifuge and absorb supernatant into buffer. 4℃ binding 3h, rotate and mix.</li>
+                        <li>After binding, Put on ice(5min), Centrifuge at 2000rpm for 1min.</li>
+                        <li>Absorb 80μL supernatant as control and remove the other supernatant, add 1 mL NPI-20 washing,
+                            upside and down to mix, still standing, Centrifuge at 2000 rpm for 1min(4℃), wash 3~5 times.
+                        </li>
+                        <li>Add 500μL NPI-250 into Beads, rotate and mix for 15min, gather supernatant, add 500 μL NPI-250 ,
+                            rotate and mix for 15min, gather supernatant again.
+                        </li>
+                    </ol>
-					<table class="table">
+                    <h2>Renaturation of the inclusion bodies</h2>
-						<tr>
-							<th style="text-align: center" colspan="4"> NPI-10 buffer（1L）pH=8.0 filtration sterilization
-							</th>
-						</tr>
-						<tr>
-							<td align="center">Reagent</td>
-							<td align="center">NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O</td>
-							<td align="center">NaCl</td>
-							<td align="center">imidazole</td>
-						</tr>
-						<tr>
-							<td align="center">Dosage</td>
-							<td align="center">6.9</td>
-							<td align="center">17.54</td>
-							<td align="center">0.68</td>
-						</tr>
-					</table>
-					<table class="table">
+                    <table class="table">
-						<tr>
+                        <tr>
-							<th style="text-align: center" colspan="4">NPI-20 buffer（1L）pH=8.0 filtration sterilization</th>
+                            <th style="text-align: center" colspan="4">Binding buffer(1L) pH=8.0</th>
-						</tr>
+                        </tr>
-						<tr>
+                        <tr>
-							<td align="center">Reagent</td>
+                            <td align="center">Reagent</td>
-							<td align="center">NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O</td>
+                            <td align="center">NaCl(500 mmol/L)</td>
-							<td align="center">NaCl</td>
+                            <td align="center">Na<sub>3</sub>PO<sub>4</sub>·12H<sub>2</sub>O(20 mmol/L)</td>
-							<td align="center">imidazole</td>
+                            <td align="center">imidazole (20 mmol/L)</td>
-						</tr>
+                        </tr>
-						<tr>
+                        <tr>
-							<td align="center">Dosage （g）</td>
+                            <td align="center">Dosage(g)</td>
-							<td align="center">6.9</td>
+                            <td align="center">29.22</td>
-							<td align="center">17.54</td>
+                            <td align="center">7.6</td>
-							<td align="center">1.36</td>
+                            <td align="center">1.36</td>
-						</tr>
+                        </tr>
-					</table>
+                    </table>
+                    <p>After cell disruption, sediment dissolves in binding buffer(8 mol/L urea)</p>
+                    <table class="table">
+                        <tr>
+                            <th style="text-align: center" colspan="5">Washing buffer(1 L)</th>
+                        </tr>
+                        <tr>
+                            <td align="center">Reagent</td>
+                            <td align="center">Tris-HCl(50 mmol/L)</td>
+                            <td align="center">EDTA(5 mmol/L)</td>
+                            <td align="center">NaCl(100 mmol/L)</td>
+                            <td align="center">Triton X-100(1%)</td>
+                        </tr>
+                        <tr>
+                            <td align="center">Dosage</td>
+                            <td align="center">100 mL</td>
+                            <td align="center">1.8612 g</td>
+                            <td align="center">5.844 g</td>
+                            <td align="center">10 mL</td>
+                        </tr>
+                    </table>
-					<table class="table">
+                    <ol>
-						<tr>
+                        <li>Induced expression</li>
-							<th style="text-align: center" colspan="4">NPI-250 buffer（1L）pH=8.0 filtration sterilization
+                        <li>Collect sediment after ultrasonication, use washing buffer including 2, 3 mol/L urea to wash
-							</th>
+                            sediment in turn. Finally, use washing buffer including 8 mol/L urea to dissolve. Centrifuge and
-						</tr>
+                            absorb supernatant, measure protein concentration and make it keep about 1 mg/mL. Dialyze these
-						<tr>
+                            supernatant using binding buffer including concentration gradient urea(6,5,4,3,2,1,0.5 and
-							<td align="center">Reagent</td>
+mol/L).
-							<td align="center">NaH2PO4·H2O</td>
+                        </li>
-							<td align="center">NaCl</td>
+                    </ol>
-							<td align="center">imidazole</td>
-						</tr>
-						<tr>
-							<td align="center">Dosage （g）</td>
-							<td align="center">6.9</td>
-							<td align="center">17.54</td>
-							<td align="center">17.0</td>
-						</tr>
-					</table>
-					<h4>Protocol</h4>
-					<ol>
-						<li>Cut tips. Add 30μL Ni 6 fast flow Beads into 1.5mL EP.</li>
-						<li>Add 1mL NPI-10 buffer, mixing wash, sedimentate at low speed and wash 3 times.</li>
-						<li>Centrifuge and absorb supernatant into buffer. 4℃ binding 3h，rotate and mix.</li>
-						<li>After binding, Put on ice(5min),Centrifuge at 2000rpm for 1min.</li>
-						<li>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.
-						</li>
-						<li>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.
-						</li>
-					</ol>
-					<h2>Renaturation of the inclusion bodies</h2>
+                    <h2>Extracting tubulin from porcine brains</h2>
+                    <ol>
+                        <li>Pick up 20 porcine brains from Beijing No.5 Meat Processing. For tubulin extraction
+                            experiment, the brains should be as fresh as possible. Take an ice box to store the brains.
+                            Avoid contact between the brains.
+                        </li>
+                        <li>While getting the brains, another student should stay in the lab and prepared the centrifuge
+                            (set one at 4℃ and another at 37℃, also pre-warm the rotor). Put electronic balance, grinder, a
+                            graduated cylinder in refrigerator and pre-warm the glycerol and PEM in 37℃. prepare the fresh
+                            ATP and GTP buffer in the morning.
+                        </li>
+                        <li>Clean the brain by tearing off the meninges and blood clots using kim wipes or by hand.</li>
+                        <li>After cleaning, weigh the brains, put the brains in the blender, then add the same volume buffer
+                            PEM(with 1 mM DTT) in it accordingly.
+                        </li>
+                        <li>Homogenate the brain for 3s, 10 times, time interval between two homogenate is 5s, in order to
+                            avoid destroy the tubulin because of high thermos.
+                        </li>
+                        <li>Pour the homogenate into a flask, incubate in 4℃ for 30 min to depolymerize microtubules.</li>
+                        <li>Pour the homogenate into tubes for Type 45Ti rotor and balance each tube.</li>
+                        <li>Centrifuge at 8000 rpm for 40min at 4℃. Filter the supernatant with 4 gauzes. Then centrifuge
+                            the filtrate at 40000 g for 40min at 4℃.
+                        </li>
+                        <li>Add 1/2 volume of warmed glycerol drop-wise with continuous shaking, mix gently but thoroughly.
+                            Add GTP (final concentration 0.1 mmol/L), MgCl<sub>2</sub> (final concentration 3 mmol/L), and
+                            EGTA (final
+                            concentration 1 mmol/L). Incubate in a 37℃ water bath for 1h, shake gently and occasionally.
+                        </li>
+                        <li>Balance each tube and centrifuge at 100000 g for 40 min at 35℃.Discard supernatant, the pellet
+                            is crude extracts. Split charge them into 50 mL centrifuge tubes, each tube contains 5 g. Snap
+                            freeze the tubulin in 15 μL aliquots in liquid nitrogen and further stored at -80℃.
+                        </li>
+                        <li>When going to do refined depuration, melt the freezing crude extract at 4℃ refrigerator on ice
+                            over night. Pop out 1 g of the pellet out of the tubes with a spatula. Put the pellets in the
+                            Dounce grinder, then add cold PEM in the tube to wash off residual pellets.
+                        </li>
+                        <li>Re-suspend the pellets with grinder, keep the grinder on ice, and grinding occasionally. After
+min, pour out the solution and rinse the grinder with cold PEM. Total re-suspended volume is
+mL.
+                        </li>
+                        <li>Add GTP (final concentration 0.1 mmol/L). Place it on ice for 1h to depolymerize. Shake it
+                            occasionally.
+                        </li>
+                        <li>Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4℃.</li>
+                        <li>Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES( pH =6.9 ),
+                            DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl<sub>2</sub> (final
+                            concentration
+mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
+                        </li>
+                        <li>Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.</li>
+                        <li>Balance each tube and centrifuge at 100000 g for 1h at 35℃.</li>
+                        <li>Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration
+.1 mmol/L). Place it on ice for 1h to depolymerize. Shake it occasionally.
+                        </li>
+                        <li>Centrifuge the depolymerized tubulin at 100000 g for 40min at 4 ℃.</li>
+                        <li>Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES ( pH= 6.9 ),
+                            DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl<sub>2</sub> (final
+                            concentration 1 mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
+                        </li>
+                        <li>Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.</li>
+                        <li>Balance each tube and centrifuge at 100000 g for 1h at 35℃.</li>
+                        <li>Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration
+.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it occasionally.
+                        </li>
+                        <li>Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4℃.</li>
+                        <li>Recover the supernatant, add equal volume polymerize buffer (containing 100 mmol/L PIPES-KOH, 2
+                            mmol/L EGTA, 2 mmol/L MgCl<sub>2</sub>, 2 mmol/L GTP and 60% glycerol).
+                        </li>
+                        <li>Incubate in a 37℃ water bath for 1 h. Adding a series concentrations of taxol.</li>
+                        <li>Balance each tube and centrifuge at 100000 g for 1 h at 35℃. The pellet is fine purified
+                            product.
+                        </li>
+                    </ol>
-					<table class="table">
+                    <h3>Preparing STM samples</h3>
-						<tr>
+                    <ol>
-							<th style="text-align: center" colspan="4">Binding buffer (1L) pH=8.0</th>
+                        <li>Use 200 # copper mesh to prepare the samples. This procedure was done in Institute of
-						</tr>
+                            Biophysics, Chinese Academy of Science. Pretreat the copper mesh with Varian Plasma Cleaner PDC-32G.</li>
-						<tr>
+                        <li>Add protein solution on copper mesh. Keep still for 1 min.</li>
-							<td align="center">Reagent</td>
+                        <li>Add 3 drops of uranyl acetate on parafilm. Mix the protein binding side with uranyl acetate
-							<td align="center">NaCl（500mmol/L）</td>
+                            thoroughly.
-							<td align="center">Na3PO4·12H2O(20mmol/L)</td>
+                        </li>
-							<td align="center">imidazole (20mmol/L)</td>
+                        <li>Store the sample carefully. Observe by transmission electron microscopy in Beijing Normal
-						</tr>
+                            University.
-						<tr>
+                        </li>
-							<td align="center">Dosage（g）</td>
+                    </ol>
-							<td align="center">29.22</td>
-							<td align="center">7.6</td>
-							<td align="center">1.36</td>
-						</tr>
-					</table>
-					<p>After cell disruption, sediment dissolves in binding buffer(8mol/L urea)</p>
-					<table class="table">
-						<tr>
-							<th style="text-align: center" colspan="5">Washing buffer（1L）</th>
-						</tr>
-						<tr>
-							<td align="center">Reagent</td>
-							<td align="center">Tris-HCl(50mmol/L)</td>
-							<td align="center">EDTA(5mmol/L)</td>
-							<td align="center">NaCl(100mmol/L)</td>
-							<td align="center">Triton X-100(1%)</td>
-						</tr>
-						<tr>
-							<td align="center">Dosage</td>
-							<td align="center">100mL</td>
-							<td align="center">1.8612g</td>
-							<td align="center">5.844g</td>
-							<td align="center">10mL</td>
-						</tr>
-					</table>
-					<ol>
+                    <h3> OD<sub>350</sub> test </h3>
-						<li>Induced expression</li>
+                    <ol>
-						<li>Collect sediment after ultrasonication, use washing buffer including 2,3mol/L urea to wash
+                        <li> Take out the tubulin monomer solution after precision purification from -80℃, and melt on the ice.
-							sediment in turn. Finally, use washing buffer including 8mol/L urea to dissolve. Centrifuge and
+                        </li>
-							absorb supernatant, measure protein concentration and make it keep about 1mg/mL. Dialyze these
+                        <li>Prepare 10 um tubulin monomer solution with the same concentration, and add isopyknic
-							supernatant using binding buffer including concentration gradient urea(6,5,4,3,2,1，0.5 and
+                            polymerization buffer (the formula is the same as the polymerization buffer used in precision
-mol/L).
+                            purification). This step finished on the ice in order to avoid the influence of the
-						</li>
+                            Spontaneous tubulin polymerization because of the high room temperature.
-					</ol>
+                        </li>
+                        <li> Prepare a series concentration gradient of taxol solution and add the
+                            tubulin polymerization system.
+                        </li>
+                        <li> Incubate for 1h at 37℃.</li>
+                        <li> Use Nanodrop UV-IS to determinate absorbance value, and set measurement parameters 280 nm, 350
+                            nm and 750 nm, but we only read at 350 nm.
+                        </li>
+                        <li> Use isopyknic mixed solution of polymerization buffer and PEM buffer to calibrate baseline,
+                            each group of samples detect three times in parallel.
+                        </li>
+                    </ol>
+                    <h2>Improvement</h2>
+                    <h3>Culture and collection</h3>
+                    <ol>
+                        <li>Use LB medium to preculture transformed media 5 ml for 12h, 200 rpm/ 37℃.</li>
+                        <li>
+                            Culture 5 mL preculture media into 100 mL TB medium for about 3h until OD<sub>600</sub>=0.4～0.6, 200 rpm/
+℃.
+                        </li>
+                        <li>Add arabinose or turn to 42℃ to induce P(3HB) expression for 72h, 220 rpm.</li>
+                        <li>Collect cells and centrifuge for 3min, 5,000 rpm.</li>
+                        <li>Remove supernatant and suspend with pure water.</li>
+                        <li>Centrifuge again for 3min, 5,000 rpm and remove its supernatant.</li>
+                    </ol>
-					<h2>Extraction tubulin from porcine brains</h2>
-					<ol>
-						<li>Picked up 20 porcine brains from Beijing No.5 Meat Processing. For tubulin extraction
-							experiment, the brains should be as fresh as possible. Take an ice box to store the brains.
-							Avoid contact between the brains.
-						</li>
-						<li>While getting the brains, another student should stay in the lab and prepared the centrifuge
-							(set one at 4℃ and another at 37℃, also pre-warm the rotor). Put electronic balance, grinder, a
-							graduated cylinder in refrigerator and pre-warm the glycerol and PEM in 37℃. prepare the fresh
-							ATP and GTP buffer in the morning.
-						</li>
-						<li>Clean the brain by tearing off the meninges and blood clots using kim wipes or by hand.</li>
-						<li>After cleaning, weigh the brains, put the brains in the blender, then add the same volume buffer
-							PEM(with 1 mM DTT) in it accordingly.
-						</li>
-						<li>Homogenate the brain for 3 s, 10 times, time interval between two homogenate is 5 s, in order to
-							avoid destroy the tubulin because of high thermos.
-						</li>
-						<li>Pour the homogenate into a flask, incubate in 4℃ for 30 min to depolymerize microtubules.</li>
-						<li>Pour the homogenate into tubes for Type 45Ti rotor and balance each tube.</li>
-						<li>Centrifuge at 8000 rpm for 40 min at 4 ℃. Filter the supernatant with 4 gauzes. Then centrifuge
-							the filtrate at 40000 g for 40 min at 4℃.
-						</li>
-						<li>Add 1/2 volume of warmed glycerol drop-wise with continuous shaking, mix gently but thoroughly.
-							Add GTP (final concentration 0.1 mmol/L), MgCl<sub>2</sub> (final concentration 3 mmol/L), and EGTA (final
-							concentration 1 mmol/L). Incubate in a 37℃ water bath for 1 h, shake gently and occasionally.
-						</li>
-						<li>Balance each tube and centrifuge at 100000 g for 40 min at 35℃.Discard supernatant, the pellet
-							is crude extracts. Split charge them into 50 ml centrifuge tubes, each tube contains 5 g. Snap
-							freeze the tubulin in 15 ul aliquots in liquid nitrogen and further stored at -80 ℃.
-						</li>
-						<li>When going to do refined depuration, melt the freezing crude extract at 4 ℃ refrigerator on ice
-							over night. Pop out 1 g of the pellet out of the tubes with a spatula. Put the pellets in the
-							Dounce grinder, then add cold PEM in the tube to wash off residual pellets.
-						</li>
-						<li>Re-suspend the pellets with grinder, keep the grinder on ice, and grinding occasionally. After
-min, pour out the solution and rinse the grinder with cold PEM. Total re-suspended volume is
-ml.
-						</li>
-						<li>Add GTP (final concentration 0.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it
-							occasionally.
-						</li>
-						<li>Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4 ℃.</li>
-						<li>Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES( pH =6.9 ),
-							DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl2 (final concentration
-mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
-						</li>
-						<li>Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.</li>
-						<li>Balance each tube and centrifuge at 100000 g for 1 h at 35℃.</li>
-						<li>Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration
-.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it occasionally.
-						</li>
-						<li>Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4 ℃.</li>
-						<li>Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES ( pH= 6.9 ),
-							DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl2 (final concentration
-mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
-						</li>
-						<li>Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.</li>
-						<li>Balance each tube and centrifuge at 100000 g for 1 h at 35℃.</li>
-						<li>Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration
-.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it occasionally.
-						</li>
-						<li>Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4℃.</li>
-						<li>Recover the supernatant, add equal volume polymerize buffer (containing 100 mmol/L PIPES-KOH, 2
-							mmol/L EGTA, 2 mmol/L MgCl2, 2 mmol/L GTP and 60% glycerol).
-						</li>
-						<li>Incubate in a 37℃ water bath for 1 h.</li>
-						<li>Balance each tube and centrifuge at 100000 g for 1 h at 35℃. The pellet is fine purified
-							product.
-						</li>
-					</ol>
+                    <h3>Extract PHB<sup>1</sup></h3>
+                    <ul>
+                        <li>Centrifuge settings: 4000RPM, 10mins.</li>
+                        <li> Scale as appropriate.</li>
+                        <li> After each centrifuge step the supernatant should be poured off.</li>
+                    </ul>
+                    <ol>
+                        <li>Resuspend precipitation in 10 mL Triton X-100(1% v/v in PBS) for 30mins at room temp.</li>
+                        <li>Centrifuge, resuspend in 10 mL PBS.</li>
+                        <li>Centrifuge, add 10 mL sodium hyperchlorite solution and incubate at 30˚C for 1 hour.</li>
+                        <li>Centrifuge, wash with 10 mL 70% EtOH.</li>
+                        <li>Allow powder to dry.</li>
+                    </ol>
+                    <p style="color: #5c5c5c;font-size: 12px;">[1]: Shahryar Shakeri, Comparison of intracellular
+                    polyhydroxybutyrate granules formation between different bacterial cell subpopulations by flow
+                    cytometry, Jundishapur Journal of Microbiology 2011.</p>
+                    <h3>Agents formula</h3>
+                    <table class="table">
+                        <tr>
+                            <th style="text-align: center" colspan="7">TB medium (1 L)</th>
+                        </tr>
+                        <tr>
+                            <td align="center">yeast extract</td>
+                            <td align="center">tryptone</td>
+                            <td align="center">K<sub>2</sub>HPO<sub>4</sub></td>
+                            <td align="center">KH<sub>2</sub>PO<sub>4</sub></td>
+                            <td align="center">Glycerol</td>
+                            <td align="center">Glucose</td>
+                            <td align="center">ddH<sub>2</sub>O</td>
+                        </tr>
+                        <tr>
+                            <td align="center">24 g</td>
+                            <td align="center">12 g</td>
+                            <td align="center">72 mM</td>
+                            <td align="center">17 mM</td>
+                            <td align="center">8 mL</td>
+                            <td align="center">2%</td>
+                            <td align="center">add to 1 L</td>
+                        </tr>
+                    </table>
-					<h2>Improvement</h2>
+                    <table class="table">
-					<h3>Culture and collection</h3>
+                        <tr>
-					<ol>
+                            <th style="text-align: center" colspan="6">PBS (1 L)</th>
-						<li>Use LB medium to preculture transformed media 5 ml for 12 hrs, 200 rpm/ 37℃.</li>
+                        </tr>
-						<li>Culture 5 mL preculture media into 100 mL TB medium for about 3 hrs until OD=0.4～0.6, 200 rpm/
+                        <tr>
-℃.
+                            <td align="center">NaCl</td>
-						</li>
+                            <td align="center">KCl</td>
-						<li>Add arabinose or turn to 42℃ to induce P(3HB) expression for 72 hrs, 220rpm.</li>
+                            <td align="center">Na<sub>2</sub>HPO<sub>4</sub>·12H<sub>2</sub>O</td>
-						<li>Collect cells and centrifuge for 3 min, 5,000 rpm.</li>
+                            <td align="center">KH<sub>2</sub>PO<sub>4</sub>·3H<sub>2</sub>O</td>
-						<li>Remove supernatant and suspend with pure water.</li>
+                            <td align="center">pH</td>
-						<li>Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.</li>
+                            <td align="center">ddH<sub>2</sub>O</td>
-					</ol>
+                        </tr>
+                        <tr>
+                            <td align="center">8 g</td>
+                            <td align="center">0.2 g</td>
+                            <td align="center">3.63 g</td>
+                            <td align="center">0.31 g</td>
+                            <td align="center">adjust to 7.4</td>
+                            <td align="center">add to 1 L</td>
+                        </tr>
+                    </table>
+                    <br />
+                    <h2>Fluorescence Detection</h2>
+                    <h3>Protein functional Test</h3>
+                    <ol>
+                        <li> Bacteria culturing and inducing for expression. </li>
+                        <li> Collect supernatant after ultrasonication. </li>
+                        <li>
+                            Concentrate the supernatant via ultra-filtering
+                            <table class="table">
+                                <tbody>
+                                    <tr>
+                                        <th style="text-align: center">Protein samples</th>
+                                        <th style="text-align: center">α-Tubulin YNE</th>
+                                        <th style="text-align: center">α-tubulin-YCE</th>
+                                        <th style="text-align: center">β-tubulin</th>
+                                        <th style="text-align: center">Aggregation buffer</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">Dosage</td>
+                                        <td align="center">75 μL</td>
+                                        <td align="center">75 μL</td>
+                                        <td align="center">150 μL</td>
+                                        <td align="center">300 μL</td>
+                                    </tr>
+                                </tbody>
+                            </table>
-					<h3>Extract PHB<sup>1</sup></h3>
+                            <table class="table">
-					<ul>
+                                <tbody>
-						<li>Centrifuge settings: 4000RPM, 10mins.</li>
+                                    <tr>
-						<li> Scale as appropriate.</li>
+                                        <th style="text-align: center">Protein samples</th>
-						<li> After each centrifuge step the supernatant should be poured off.</li>
+                                        <th style="text-align: center">α-tubulin-YCE</th>
-					</ul>
+                                        <th style="text-align: center">β-tubulin- YNE</th>
-					<ol>
+                                        <th style="text-align: center">Aggregation buffer</th>
-						<li>Resuspend precipitation in 10 mL Triton X-100(1% v/v in PBS) for 30mins at room temp.</li>
+                                    </tr>
-						<li>Centrifuge, resuspend in 10 mL PBS.</li>
+                                    <tr>
-						<li>Centrifuge, add 10 mL sodium hyperchlorite solution and incubate at 30˚C for 1 hour.</li>
+                                        <td align="center">Dosage</td>
-						<li>Centrifuge, wash with 10 mL 70% EtOH.</li>
+                                        <td align="center">150 μL</td>
-						<li>Allow powder to dry.</li>
+                                        <td align="center">150 μL</td>
-					</ol>
+                                        <td align="center">300 μL</td>
-					<p style="color: #5c5c5c;font-size: 12px;">[1]: Shahryar Shakeri, Comparison of intracellular polyhydroxybutyrate granules formation between different bacterial cell subpopulations by flow cytometry, Jundishapur Journal of Microbiology 2011.</p>
+                                    </tr>
-					<h3>Agents formula</h3>
+                                </tbody>
-					<table class="table">
+                            </table>
-						<tr>
-							<th style="text-align: center" colspan="7">TB medium (1L)</th>
-						</tr>
-						<tr>
-							<td align="center">yeast extract</td>
-							<td align="center">tryptone</td>
-							<td align="center">K2HPO4</td>
-							<td align="center">KH2PO4</td>
-							<td align="center">Glycerol</td>
-							<td align="center">Glucose</td>
-							<td align="center">ddH2O</td>
-						</tr>
-						<tr>
-							<td align="center">24 g</td>
-							<td align="center">12 g</td>
-							<td align="center">72 mM</td>
-							<td align="center">17 mM</td>
-							<td align="center">8 mL</td>
-							<td align="center">2%</td>
-							<td align="center">add to 1 L</td>
-						</tr>
-					</table>
-					<table class="table">
+                        </li>
-						<tr>
+                        <li> Mixed the protein samples and add proper GTP, taxol to 200uM. </li>
-							<th style="text-align: center" colspan="6">PBS (1L)</th>
+                        <li> Use absolute recording spectrofluorometer with 514nm excitation. </li>
-						</tr>
+                        <li> Record light intensity in 520nm-530nm wavelength emission. </li>
-						<tr>
+                    </ol>
-							<td align="center">NaCl</td>
-							<td align="center">KCl</td>
-							<td align="center">Na2HPO4·12H2O</td>
-							<td align="center">KH2PO4·3H2O</td>
-							<td align="center">pH</td>
-							<td align="center">ddH2O</td>
-						</tr>
-						<tr>
-							<td align="center">8 g</td>
-							<td align="center">0.2 g</td>
-							<td align="center">3.63 g</td>
-							<td align="center">0.31g</td>
-							<td align="center">adjust to 7.4</td>
-							<td align="center">add to 1 L</td>
-						</tr>
-					</table>
-				</article>
+                    <h3>Taxol-concentration based assay</h3>
-			</div>
-		</div>
+                    <ol>
-	</div>
+                        <li> Bacteria culturing and inducing for expression </li>
+                        <li> Collect supernatant after ultrasonication </li>
+                        <li>
+                            Concentrate the supernatant via ultra-filtering
+                            <table class="table">
+                                <tbody>
+                                    <tr>
+                                        <th style="text-align: center">Protein samples</th>
+                                        <th style="text-align: center">α-Tubulin YNE</th>
+                                        <th style="text-align: center">α-tubulin-YCE</th>
+                                        <th style="text-align: center">β-tubulin</th>
+                                        <th style="text-align: center">Aggregation buffer</th>
+                                    </tr>
+                                    <tr>
+                                        <td align="center">Dosage</td>
+                                        <td align="center">75 μL</td>
+                                        <td align="center">75 μL</td>
+                                        <td align="center">150 μL</td>
+                                        <td align="center">300 μL</td>
+                                    </tr>
+                                </tbody>
+                            </table>
+                        </li>
+                        <li>
+                            Mixed the protein samples and add taxol respectively as the following table:
+                            <table class="table">
+                                <tbody>
+                                    <tr>
+                                        <td align="center">Taxol concentration (μM)</td>
+                                        <td align="center">0</td>
+                                        <td align="center">0.5</td>
+                                        <td align="center">5</td>
+                                        <td align="center">25</td>
+                                        <td align="center">50</td>
+                                        <td align="center">100</td>
+                                        <td align="center">250</td>
+                                        <td align="center">500</td>
+                                    </tr>
+                                </tbody>
+                            </table>
+                        </li>
+                        <li> Use absolute recording spectrofluorometer with 514nm excitation </li>
+                        <li> Record light intensity in 520nm-530nm wavelength emission. </li>
+                    </ol>
+                </article>
+            </div>
+        </div>
+    </div>
 </html>

Difference between revisions of "Team:BNU-China/Protocol"

Latest revision as of 02:16, 20 October 2016

Protocol

Cloning

PCR

Reaction system:

Process:

Fusion PCR：

The system of step 2:

Electrophoresis---Gel Purification

Material:

Protocol:

Digestion

Ligation

LR reaction

1. Entry linearization

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

Transformation

Material:

Protocol:

Protein expression

Detection

SDS-PAGE

Materials

Protocol

Western Blot

System

Protocol

Ni-beads protein purification

Protocol

Renaturation of the inclusion bodies

Extracting tubulin from porcine brains

Preparing STM samples

OD350 test

Improvement

Culture and collection

Extract PHB1

Agents formula

Fluorescence Detection

Protein functional Test

Taxol-concentration based assay

OD₃₅₀ test

Extract PHB¹