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Protocol

Miniprep

Materials

Buffer 1	250μL
Buffer 2	250μL
Buffer 3	350μL
Buffer 4	500μL
Buffer 5	700μL
Distilled water	100μL
Sample

Procedure

Suspend the sample in buffer 1 (1.5 ml sample tube)
Add buffer 2 to the sample and mix it
Add buffer 3 to the sample and mix it
Centrifuge for 5 minutes at 13,000 rpm and apply the supernatants to the column
Add buffer 4 and centrifuge for 1 minute at 13,000 rpm then throw away the filter paper
Add buffer 5 and centrifuge for 1 minute at 13,000 rpm then throw away the filter paper
Centrifuge again for 1 minute at 13,000 rpm
Place the column on a new sample tube and add 100 μL distilled water
Centrifuge for 1 minute at 6,000 rpm

Recipes for Buffer

Buffer 1 (Suspension Buffer)	50 mM Tris-HCl and 10 mM EDTA, pH 8.0 (25°C) 50 μg/ml RNase A
Buffer 2 (Lysis Buffer)	0.2 M NaOH and 1% SDS
Buffer 3 (Neutralization and Binding Buffer)	4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2
Buffer 4 (Wash Buffer)	5 M guanidine hydrochloride, 20 mM Tris-HCl, pH 6.6 (25°C) [final conentrations after addition of ethanol] with 38% ethanol
Buffer 5 (Wash Buffer)	20 mM NaCl, 2 mM Tris-HCl, pH 7.5 (25°C) [final conentrations after addition of ethanol] with 80% ethanol

PCR-1

Materials

Total	100μL
Buffer for KOD-FX-NEO	50μL
dNTP	20μL
Primer mix	1.0μL
Template DNA	0.5μL
KOD-FX-NEO (TOYOBO)	2.0μL
H2O	26.5μL

Procedure

Add buffer, dNTP, primer mix, Template DNA, KOD-FX-NEO and distilled water to an Eppendorf tube and mix
Place the PCR tubes into the PCR machine and set the program

PCR Profile for KOD-FX-NEO

Predenature	Denature	Annealing	Extension	Final Extension
98.0°C KOD-FX-NEO	98.0°C	51.0°C	68.0°C	68.0°C
2 minutes	10 seconds	30 seconds	1 minute	2 minutes
1 cycle	30 cycles			1 cycle

PCR Primer Design

Lot.No	Oligo	Base Number	Sequence
T14F190235	pGEX6P-2F1	30	CACACAGGAACTAGTATTCATGTCCCCTAT
T14F190236	pGEX6P-2F2	30	GGATCTGGAAGTTCTGTTCCACTAGTCCCT
T14F190237	pGEX6P-2R	29	TCATCACCGAAACGCTCGAGGCAGATCGT

PCR-2

Materials

Total	100μL
Taq Buffer	50μL
dNTP	20μL
Primer mix	1.0μL
Template DNA	0.5μL
Taq polymerase	2.0μL
H2O	26.5μL

Procedure

Add buffer, dNTP, primer mix, Template DNA, Taq polymerase and distilled water to an Eppendorf tube and mix
Place the PCR tubes into the PCR machine and set the program

PCR Profile for KOD-FX-NEO

Predenature	Denature	Annealing	Extension	Final Extension
98.0°C Taq polymerase	95.0°C	53.0°C	72.0°C	72.0°C
30 seconds	30 seconds	45 seconds	2 minutes	1 minute
1 cycle	30 cycles			1 cycle

PCR Primer Design

Lot.No	Oligo	Base Number	Sequence
T14H280166	Terpinene3-suffix-Spe1	30	CAGTCAGTCACGAACTAGTTTAAGGATGGG
T14H280167	Terpinene3-prefix-Xba1	30	CCGGGTTCTAGATGGCTGCATCCACTACTT
T14H180317	Terpinene3-mutagenesis-FP	28	TTGGAGTTCTTGCAGGATTCTCCCGATA
T14H180318	Terpinene3-mutagenesis-RP	26	GGCCTCCTTTGTTATTGGATTTGCTG

Restriction Digest

Materials

DNA sample	6μL
Buffer	1μL
Restriction Enzyme	0.5μL
Distilled water	2.5μL

Procedure

Add all materials to a tube and mix them gently
Incubate at 37˚C for the enzyme reaction for 15 minutes

AGE

Materials

Sample	5μL
Agarose gel
2X Loading Buffer Triple Dye (NIPPON GENE)	5μL
1X TAE Buffer

Procedure

Set an agarose gel on an electrophoresis chamber
Add 1X TAE buffer to the electrophoresis chamber
Note: Do not generate bubbles under the gel
Add 2X loading buffer to electrophoresis samples
Apply samples on agarose gel wells
Set an appropriate voltage (100 V) and run the electrophoresis
Stop the electrophoresis when the BPB reaches 2/3 of the gel
Soak the gel in EtBr (ethidium bromide) solution and dye it for 20 minutes
Place plastic cooking wrap on the trans-illuminator and irradiate UV to the gel on the wrap
Take photographs of the gel by using a trans-illuminator

Agarose Concentration Versus Optical Range of DNA Size

Agarose Concentration (%)	DNA (kbp)
0.3	5-60
0.6	1-20
1.0	0.3-7
1.5	0.2-4
2.0	0.1-2

DNA Refinement 1

Materials

DNA sample	200μL
distilled H2O	400μL
CH3COONa	50μL
Phe/Chl	700μL
Isopropyl alcohol	750μL
75% Ethanol	1000μL

Procedure

Add materials (DNA, H2O, CH3COONa, and Phe/Chl) to sample tube and vortex
Centrifuge at 14,500 rpm for 10 minutes at room temperature
Transfer the supernatant to a new sample tube
Add 750 μL of isopropyl alcohol to the sample tube and vortex
Centrifuge at 14,500 rpm for 10 minutes at 4°C
Remove isopropyl alcohol gently so that the pellet will not break
Add 1,000 μL of 75% ethanol and centrifuge at 14,500 rpm for 5 minutes at 4°C
Dry it by aspirator for 10 minutes
Add 25 μL of distilled H2O and mix

Note

Phe/Chl composition Phenol:Chloroform:Isoamyl alcohol=25:24:1

DNA Refinement 2

Materials

sample	30μL
Bacterial Alkaline Phosphatase (BAP)	1μL
Low melting agarose gel
6×Loading Buffer Double Dye	5μL
Distilled water	200μL
Phenol
Phe/Chl
Isopropyl alcohol	750μL
5M CH3COONa	50μL
70% Ethanol	1000μL

Procedure

Add BAP to vector and dephosphorize for 30 minutes at 37°C
Run the electrophoresis with agarose gel
Cut out the targeted band and mix with 200 μL of distilled water in a tube
Heat the sample for 5 minutes at 65°C
Add the same amount of phenol as the sample and vortex
Centrifuge at 14,500 rpm for 10 minutes at room temperature and apply the supernatant to the new sample tube
Add the same amount of Phe/Chl as the sample and vortex
Centrifuge at 14,500 rpm for 5 minutes at room temperature and apply the supernatant to the sample tube
Add 750 μL of Isopropyl alcohol and 50 μL of CH3COONa and centrifuge at 14,500rpm for 10 minutes at 4°C
Rinse with 70% ethanol at 14,500 rpm 5 minutes at 4°C
Dry it for 15 minutes

Ligation

Materials

DNA sample	5μL
DNA ligase (Ligation High from Nippon gene)	5μL

Procedure

Mix DNA sample and DNA ligase in a sample tube gently
Ligation for 15 minutes at room temperature

Transformation (E. coli)

Materials

DNA Sample	10μL
Competent cell	50μL

Procedure

Thaw the competent cells on ice
Add 10 μL of sample into thawed competent cells
Cool the sample tube, which contains competent cells and DNA samples, with ice for 1 hour, then Heat shock the cells by immersion in pre-hearted water bath at 42°C for 30 seconds
Place the tube on ice for 2 minutes to cool it down
At a clean bench, add 1.0 ml of LB medium into the tube and suspend it
Incubate the tube at 37°C for 35 minutes
Harvest the cells by centrifuge
Spread the transformed competent cells onto the agar plate and incubate it at 37°C overnight

Transformation (S. cerevisiae)

Materials

Sample	5μL
Competent cells	95μL
YPD plate containing G418

Procedure

Cool down a cuvette for 5 minutes on ice
Harvest the yeast cells by centrifuge at 4 °C
Suspend the cells in 20 ml of cold 1M sorbitol and harvest by centrifuge at 4 °C (2times)
Suspend the cells in 500μL of cold 1M sorbitol
Mix DNA sample (5 μL) and the cell suspension (95 μL) in a cold cuvette
Electroporate under the following conditions: 200 Ω, 25 μF and 1.5 kV
Immediately inoculate on YPD plates containing G418
Incubate at 30 °C for 2 days

Preculture

Materials

Sample (E. coli cells )
Medium (LB medium with appropriate antibiotics)	20ml

Procedure

Scrape samples from the agar plate and inoculate them in liquid media
Cultivate overnight (37℃, 120 rpm)

Main Culture

Materials

LB medium	100ml
IPTG	10μL
Sample	500μL

Procedure

Add samples to LB medium and cultivate (37°C, 120 rpm) until reaching a cell density of OD600=0.5
Add IPTG (10 μL) to the medium (25 ml)
Further cultivation for 3 hours

Protein Extraction (E. coli)

Materials

Sample
FastBreak Cell Lysis Reagent, 10X (Promega)
50mM potassium phosphate buffer (=pH6.8)
SDS sample buffer

Procedure

Separate samples into two and harvest by centrifuge
Add potassium phosphate buffer, then mix and remove medium completely
Add FastBreak Cell Lysis Reagent, 10X at the ratio of FastBreak Buffer Cell Lysis Reagent,10X: Samples=1:9 and extract protein for 15 minutes at room temperature

Protein Extraction (S. cerevisiae)

Materials

Glass Beads
50 mM potassium phosphate buffer (pH6.8)
SDS sample buffer

Procedure

Harvest yeast cells by centrifuge at 3,000 rpm for 4 minutes at room temperature
Wash cells once with potassium phosphate buffer
Cool down the sample tube on ice for 1 minute
Add the same amount of glass beads as the sample then add 20 μL of potassium phosphate buffer and mix
Disrupt the cells by FastPrep 24 (MP Biochemicals) for 45 seconds at speed 4.5
Cool down the sample tube on ice for 1 minute
Add 80 µl of potassium phosphate buffer, cool it down and centrifuge for 5 minutes at 14,500 rpm at 4°C
Transfer 40 µl of the supernatant and mix with 8 µl of SDS sample buffer
Treat the sample with heat for 5 minutes at 100˚C

Colony Sweep

Materials

Sample
Phenol/Chloroform water-saturated solution (Phe/Chl)
Cracking solution 3% w/v SDS, 50 mM Tris-base, pH12.6

Procedure

Dispense cracking solution (50 μL) each into sample tubes
Collect the sample and suspend it into cracking solution
Incubate at 65°C for 10 minutes
Add Phe/Chl and BPB pigment and vortex
Centrifuge at 14,000 rpm for 5 minutes
Apply the samples (upper layer) to agarose gel with loading dye
Check the bands by agar gel electrophoresis

SDS-PAGE

Materials

Sample
Separating Gel
Stacking Gel

Procedure

Place stacking gel on separating gel
Rinse the wells of gel with distilled water
Load prepared samples into wells
Run the electrophoresis (25 mA for 75 min)
Check the bands with CBB stain

Separating Gel

Acylamide (%)	7.5	10	12.5	15	17.5
MilliQ H2O (ml)	3.89	3.22	2.55	1.87	1.2
Acrylamide/Bis-acrylamide (30%/0.8% w/v)	1.99	2.7	3.38	4.04	4.7
1.5M Tris-HCl(pH8.8) (ml)	2	2	2	2	2
10% (w/v)SDS (μL)	80	80	80	80	80
10% (w/v) ammonium persulfate (AP) (μL)	27	27	27	27	27
TEMED (μL)	4	4	4	4	4

Stacking Gel

MilliQ H2O (ml)	2.89
30% (w/v) acrylamide (ml)	0.79
0.5M Tris-HCl(pH6.8) (ml)	1.25
10% SDS (μL) (ml)	50
10% APS (μL) (ml)	17
TEMED (μl) (ml)	5

Western Blotting

Materials

BufferⅠ	0.3 M Tris base, 20% MetOH (pH 10.9)
BufferⅡ	25 mM Tris base, 20% MetOH
BufferⅢ	25 mM Tris base, 0.05 mM Boric acid, 20% MetOH (pH 9.5)
PBS	137 mM NaCl, 8.1 mM Na2HPO4 (12H2O), 2.68 mM KCl, 1.47 mM KH2PO4
PBS-S	PBS including 0.1% skim milk
PBS-T	PBS including 0.05% Tween20
PBS-TS	PBS including 0.1% skim milk & 0.05% Tween20
PonceauS (Sigma)
PVDF membrane (Immobilon Transfer membrane by Millipore)	1 sheet
Whatman paper (Whatman 3MM)
Hybridization bag (Cosmo Bio)
Peroxidase Stain Kit (Nacalai Tesque)
Anti-GST antibody conjugated with HRP (Wako)

Procedure

Cut the gel in appropriate size
Soak gel in buffer III and shake gently for 15 minutes
Soak the membrane on methanol then transfer it in buffer III
Soak Whatman papers (all in the same size) in Buffer I (2 sheets), Buffer II (1 sheet) and Buffer III (3 sheets) respectively
Wet the surface of the blotter
Put items in the following order: Buffer III paper, gel, membrane, Buffer II paper and Buffer I paper
Blot at the constant current of membrane's area ×2.5 mA for 20 minutes
Dye the membrane with Ponceau S for 5 minutes, rinse it with distilled water and scan it
Shake and wash with PBS-TS (3 minutes ×3times)
The membrane was incubated with anti-GST antibody containing HRP in PBS-S at room temperature for 1 hour
Wash with PBS-T twice (5 minutes/10 minutes)
Wash with PBS twice (5 minutes/5 minutes)
Stain the membrane using Peroxidase Stain Kits
Scan it

LB Medium

Materials

Tryptone	final concentration: 1%(w/v)
Yeast Extract	final concentration: 0.5%(w/v)
NaCl	final concentration: 1%(w/v)
5M NaOH

Procedure

Dissolve Tryptone (1.0 g), Yeast Extract (500 mg) and NaCl (1.0 g) in distilled water (90 ml)
Adjust pH to 7.0 by adding 20 μL of 5M NaOH
Fill up to 100 ml with distilled water
Autoclave

Note

To make agar plate, add agar powder at the final concentration of 2 %(w/v) in Procedure 2

YPD Medium

Materials

Peptone	final concentration: 2%(w/v)
Yeast extract	final concentration: 1%(w/v)
Glucose	final concentration: 2%(w/v)

Procedure

Dissolve peptone (2.0 g), yeast extract (1.0 g) and glucose (2.0 g) to distilled water (90 ml)
Fill up to 100 ml with distilled water
Sterilize by autoclave

Note

To make agar plate, add agar powder (final concentration: 2.0%) at procedure 1.

SD Medium

Materials

Yeast nitrogen base	0.67%(w/v)
Glucose	2% (w/v)
Adenine	0.004% (w/v)
Histidine	0.002% (w/v)
Leucine	0.006% (w/v)
Lysine	0.003% (w/v)
Methionine	0.002% (w/v)
Tryptophan	0.004% (w/v)

Procedure

Dissolve yeast nitrogen base (6.7 g), adenine (40 mg), histidine (20 mg), leucine (60 mg), lysine (30mg), tryptophan (40mg) and methionine (20mg) into 40 ml of distilled H2O, and then fill up to 50 ml with H2O.
Dissolve glucose (20.0 g) into 40 ml of distilled H2O, and then fill up to 50 ml with H2O.
Sterilize each solution by autoclave.
Mix solutions after cooling it down.

Note

Autoclave glucose and agar plate separately from other materials

No. 1 Inverse PCR

Prepare PCR primers (10 pmol/μL) and template plasmid (50 ng/μL)

Prepare the PCR reaction mixture as follows:

Total	50μL
PCR grade water	35μL
10×Buffer for iPCR	5μL
2 mM dNTPs	5μL
Primer A (10 pmol/μL)	1.5μL
Primer B (10 pmol/μL)	1.5μL
Plasmid Template DNA (50ng/μL)	1μL
KOD-Plus-DNA Polymerase	1μL

Perform PCR reaction as follows: PCR Profile for KOD-FX-NEO

94.0°C 98.0°C 68.0°C 4.0°C

2 minutes 10 seconds 7 minutes Hold

1 cycle 20 cycles 1 cycle

[Optional] The control reaction is as follows:

Total	50μL
PCR grade water	35μL
10×Buffer for iPCR	5μL
2 mM dNTPs	5μL
Control Primer #1（10 pmol/μL）	1.5μL
Control Primer #2（10 pmol/μL）	1.5μL
Control Plasmid pAK119M （50 ng/μL）	1μL
KOD -Plus- (1 U/μL)	1μL

94.0°C	98.0°C	98.0°C
2 minutes	10 seconds	5 minutes
1 cycle	5 cycles

No. 2 Dpn1 digestion of the template plasmid

Add 2 μL of Dpn1 restriction enzyme (10 U/μL) to 50 μL PCR reaction from step 1.
Gently and thoroughly mix each reaction mixture by pipetting
Spin down the reaction mixture and incubate at 37°C for 1 hr to digest the template plasmid

（Optional） Gel electrophoresis
To confirm PCR amplification, we recommend agarose gel electrophoresis analysis. However, in the case of 5 or less cycles of amplification, it may be only possible to detect a faint band or no band at all. If only a faint or no band is detected, please proceed to the next step.

No. 3 Self-ligation of the PCR Product

Prepare the kination/ligation reaction mixture using a fresh tube as follows:

Total Volum	15μL
Dpn1- treated PCR product	2μL
PCR grade water	7μL
Ligation high	5μL
T4 Polynucleotide Kinase	1μL

Gently and thoroughly mix each reaction mixture by pipetting
Centrifuge the reaction mixture and incubate each reaction at 16°C for 1 hr

No. 4 Transformation

Standard E. coli strains can be used as the transformation host. Both electrocompetent and chemically competent E. coli cells can be used. The following protocol is a standard protocol using chemically prepared competent E. coli cells.

Gently thaw the competent cells （100 μL） on ice.
Add 10 μL of reaction mixture from Step 3 to the competent cells, swirl gently and incubate for 30 min on ice.
Incubate for 30 seconds at 42°C and then place on ice for 2 min
Add 900 μL of SOC medium and incubate at 37°C for 1 hr with shaking.
Plate appropriate volumes (10~200 μL) of transformed E. coli solution on agar plates containing the appropriate antibiotic for the plasmid vector.
Incubate the plates at 37°C for ~16 hr.

No. 5 Analysis of Transfomants

A typical mutagenesis efficiency of this kit is greater than 80%. Therefore, analysis of 4-8 clones is typically appropriate for sequence verification.

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+  <strong>Materials</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>Buffer 1
+     </td>
+    <td>250μL</td>
+  </tr>
+  <tr>
+    <td>Buffer 2
+    </td>
+    <td>250μL</td>
+  </tr>
+  <tr>
+    <td>Buffer 3</td>
+    <td>350μL</td>
+  </tr>
+  <tr>
+    <td>Buffer 4</td>
+    <td>500μL</td>
+  </tr>
+  <tr>
+    <td>Buffer 5</td>
+    <td>700μL</td>
+  </tr>
+  <tr>
+    <td>Distilled water</td>
+    <td>100μL</td>
+  </tr>
+  <tr>
+    <td>Sample</td>
+    <td></td>
+  </tr>
+    </table>
+</div>
+<!--materials終わり-->
+  <br>
+<!--procedure始まり-->
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Suspend the sample in buffer 1 (1.5 ml sample tube)
      </li>
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+     <li>Add buffer 2 to the sample and mix it
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-     <li class="menuimg">
+     <li>Centrifuge for 5 minutes at 13,000 rpm and apply the supernatants to the column
-    <a href="/Team:KIT-Kyoto/Test/Safety">
-    safety
-    </a>
      </li>
-     <li class="menuimg">
+     <li>Add buffer 4 and centrifuge for 1 minute at 13,000 rpm then throw away the filter paper
-    <a href="/Team:KIT-Kyoto/Test/Attributions">
-    attributions
-    </a>
      </li>
+    <li>Add buffer 5 and centrifuge for 1 minute at 13,000 rpm then throw away the filter paper
+    </li>
+    <li>Centrifuge again for 1 minute at 13,000 rpm
+    </li>
+    <li>Place the column on a new sample tube and add 100 μL distilled water
+    </li>
+    <li>Centrifuge for 1 minute at 6,000 rpm
+    </li>
    </ul>
+<!--procedure終わり-->
+  <br>
+<!--Recipes for Buffer始まり-->
+  <strong>Recipes for Buffer</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>Buffer 1 (Suspension Buffer)
+    </td>
+    <td>50 mM Tris-HCl and 10 mM EDTA, pH 8.0 (25°C) 50 μg/ml RNase A
+    </td>
+  </tr>
+  <tr>
+    <td>Buffer 2 (Lysis Buffer)
+    </td>
+    <td>0.2 M NaOH and 1% SDS
+    </td>
+  </tr>
+  <tr>
+    <td>Buffer 3 (Neutralization and Binding Buffer)
+    </td>
+    <td>4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2
+    </td>
+  </tr>
+  <tr>
+    <td>Buffer 4 (Wash Buffer)
+    </td>
+    <td>5 M guanidine hydrochloride, 20 mM Tris-HCl, pH 6.6 (25°C) [final conentrations after addition of ethanol] with 38% ethanol</td>
+  </tr>
+  <tr>
+    <td>Buffer 5 (Wash Buffer)
+    </td>
+    <td>20 mM NaCl, 2 mM Tris-HCl, pH 7.5 (25°C) [final conentrations after addition of ethanol] with 80% ethanol</td>
+  </tr>
+    </table>
+<!--Recipes for Buffer終わり-->
+  <br>
+  </p>
+<!--本文終わり-->
+</div>
    </div>
-   <!--サイドメニュー終わり-->
+   </div>
+<!--Miniprepプロトコル終わり-->
+<!--PCR-1-->
-  <!--メインコンテンツ-->
-  <div id="container">
-  <div class="main-contents">
-  <h1>Protocol</h1>
-<!--PCR-->
    <div class="accordion">
    <div class="scroll">
    </div>
-   <h2><a href="javascript:void(0);">PCR</a></h2>
+   <h2><a href="javascript:void(0);">PCR-1</a></h2>
    <div class="ac">
    <p class="sentence">
    <strong>Materials</strong>
+<div class="m_table">
    <table class="materials">
     <tr>
@@ Line 160: / Line 293: @@
     </tr>
    </table>
+</div>
    <br>
    <strong>Procedure</strong>
@@ Line 169: / Line 303: @@
    <br>
 <strong>PCR Profile for KOD-FX-NEO</strong>
+<div class="m_table">
    <table class="materials">
     <tr>
@@ Line 188: / Line 323: @@
      <td>10 seconds</td>
      <td>30 seconds</td>
-     <td>1 minutes</td>
+     <td>1 minute</td>
      <td>2 minutes</td>
     </tr>
     <tr>
      <td>1 cycle</td>
-     <td>30 cycles</td>
+     <td colspan="3" style="background-color:rgba(0,0,0,0.1);">30 cycles</td>
-    <td></td>
-    <td></td>
      <td>1 cycle</td>
     </tr>
    </table>
+<!--materials始まり-->
+<br>
+  <strong>PCR Primer Design</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <th>Lot.No
+    </th>
+    <th>Oligo</th>
+    <th>Base Number</th>
+    <th>Sequence</th>
+  </tr>
+  <tr>
+    <td>T14F190235
+    </td>
+    <td>pGEX6P-2F1</td>
+    <td>30</td>
+    <td>CACACAGGAACTAGTATTCATGTCCCCTAT</td>
+  </tr>
+  <tr>
+    <td>T14F190236
+    </td>
+    <td>pGEX6P-2F2</td>
+    <td>30</td>
+    <td>GGATCTGGAAGTTCTGTTCCACTAGTCCCT</td>
+  </tr>
+  <tr>
+    <td>T14F190237
+    </td>
+    <td>pGEX6P-2R</td>
+    <td>29</td>
+    <td>TCATCACCGAAACGCTCGAGGCAGATCGT</td>
+  </tr>
+    </table>
+</div>
+<!--PCR Primer Design終わり-->
+  <br>
+</div>
    <br>
    </p>
    </div>
    </div>
-<!--PCR終わり-->
+<!--PCR-1終わり-->
+<!--PCR-2-->
+  <div class="accordion">
+  <div class="scroll">
+  </div>
+  <h2><a href="javascript:void(0);">PCR-2</a></h2>
+  <div class="ac">
+  <p class="sentence">
+  <strong>Materials</strong>
+<div class="m_table">
+  <table class="materials">
+   <tr>
+    <td>Taq Buffer</td>
+    <td>50μL</td>
+   </tr>
+   <tr>
+    <td>dNTP</td>
+    <td>20μL</td>
+   </tr>
+   <tr>
+    <td>Primer mix</td>
+    <td>1.0μL</td>
+   </tr>
+   <tr>
+    <td>Template DNA</td>
+    <td>0.5μL</td>
+   </tr>
+   <tr>
+    <td>Taq polymerase</td>
+    <td>2.0μL</td>
+   </tr>
+   <tr>
+    <td>H2O</td>
+    <td>26.5μL</td>
+   </tr>
+   <tr>
+    <th>Total</th>
+    <th>100μL</th>
+   </tr>
+  </table>
+</div>
+  <br>
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Add buffer, dNTP, primer mix, Template DNA, Taq polymerase and distilled water to an Eppendorf tube and mix
+    </li>
+    <li>Place the PCR tubes into the PCR machine and set the program
+  </ul>
+  <br>
+<strong>PCR Profile for KOD-FX-NEO</strong>
+<div class="m_table">
+  <table class="materials">
+   <tr>
+    <th>Predenature</th>
+    <th>Denature</th>
+    <th>Annealing</th>
+    <th>Extension</th>
+    <th>Final Extension</th>
+   </tr>
+   <tr>
+    <td>98.0°C Taq polymerase</td>
+    <td>95.0°C</td>
+    <td>53.0°C</td>
+    <td>72.0°C</td>
+    <td>72.0°C</td>
+   </tr>
+   <tr>
+    <td>30 seconds</td>
+    <td>30 seconds</td>
+    <td>45 seconds</td>
+    <td>2 minutes</td>
+    <td>1 minute</td>
+   </tr>
+   <tr>
+    <td>1 cycle</td>
+    <td colspan="3" style="background-color:rgba(0,0,0,0.2);">30 cycles</td>
+    <td>1 cycle</td>
+   </tr>
+  </table>
+</div>
+  <br>
+<!--materials始まり-->
+<br>
+  <strong>PCR Primer Design</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <th>Lot.No
+    </th>
+    <th>Oligo</th>
+    <th>Base Number</th>
+    <th>Sequence</th>
+  </tr>
+  <tr>
+    <td>T14H280166
+    </td>
+    <td>Terpinene3-suffix-Spe1</td>
+    <td>30</td>
+    <td>CAGTCAGTCACGAACTAGTTTAAGGATGGG</td>
+  </tr>
+  <tr>
+    <td>T14H280167
+    </td>
+    <td>Terpinene3-prefix-Xba1</td>
+    <td>30</td>
+    <td>CCGGGTTCTAGATGGCTGCATCCACTACTT</td>
+  </tr>
+  <tr>
+    <td>T14H180317
+    </td>
+    <td>Terpinene3-mutagenesis-FP</td>
+    <td>28</td>
+    <td>TTGGAGTTCTTGCAGGATTCTCCCGATA</td>
+  </tr>
+  <tr>
+　　<td>T14H180318</td>
+    <td>Terpinene3-mutagenesis-RP</td>
+    <td>26</td>
+    <td>GGCCTCCTTTGTTATTGGATTTGCTG</td>
+    </table>
+</div>
+<!--PCR Primer Design終わり-->
+  <br>
+  </p>
+  </div>
+  </div>
+<!--PCR-2終わり-->
+<!-- Restriction Digest プロトコル -->
+  <div class="scroll">
+  </div>
+ <div class="accordion">
+<!--タイトル-->
+  <h2><a href="javascript:void(0);">Restriction Digest</a></h2>
+<!--タイトル終わり-->
+  <div class="ac">
+<!--本文-->
+  <p class="sentence">
+<!--materials始まり-->
+  <strong>Materials</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>DNA sample
+    </td>
+    <td>6μL</td>
+  </tr>
+  <tr>
+    <td>Buffer
+    </td>
+    <td>1μL</td>
+  </tr>
+  <tr>
+    <td>Restriction Enzyme</td>
+    <td>0.5μL</td>
+  </tr>
+  <tr>
+    <td>Distilled water
+    </td>
+    <td>2.5μL</td>
+  </tr>
+  </table>
+</div>
+<!--materials終わり-->
+  <br>
+<!--procedure始まり-->
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Add all materials to a tube and mix them gently    </li>
+    <li>Incubate at 37˚C for the enzyme reaction for 15 minutes     </li>
+  </ul>
+<!--procedure終わり-->
+  <br>
+  <br>
+  </p>
+<!--本文終わり-->
+  </div>
+  </div>
+<!--Restriction Digestプロトコル終わり-->
 <!--AGE-->
@@ Line 247: / Line 599: @@
      <li>Apply samples on agarose gel wells
      </li>
-     <li>Set an appropriate voltage (100v) and run the electrophoresis
+     <li>Set an appropriate voltage (100 V) and run the electrophoresis
      </li>
      <li>Stop the electrophoresis when the BPB reaches 2/3 of the gel
      </li>
-     <li>Soak the gel in EtBr (ethidium bromide) and dye it for 20 minutes
+     <li>Soak the gel in EtBr (ethidium bromide) solution and dye it for 20 minutes
      </li>
      <li>Place plastic cooking wrap on the trans-illuminator and irradiate UV to the gel on the wrap
@@ Line 260: / Line 612: @@
    <br>
     <strong>Agarose Concentration Versus Optical Range of DNA Size</strong>
+<div class="m_table">
    <table class="materials">
     <tr>
@@ Line 287: / Line 640: @@
     </tr>
    </table>
+</div>
    <br>
    </p>
@@ Line 292: / Line 646: @@
 </div>
 <!--AGE終わり-->
+<!-- DNA Refinement 1プロトコル -->
+  <div class="scroll">
+  </div>
+ <div class="accordion">
+<!--タイトル-->
+  <h2><a href="javascript:void(0);">DNA Refinement 1</a></h2>
+<!--タイトル終わり-->
+  <div class="ac">
+<!--本文-->
+  <p class="sentence">
+<!--materials始まり-->
+  <strong>Materials</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>DNA sample
+    </td>
+    <td>200μL</td>
+  </tr>
+  <tr>
+    <td>distilled H2O
+    </td>
+    <td>400μL</td>
+  </tr>
+  <tr>
+    <td>CH3COONa</td>
+    <td>50μL</td>
+  </tr>
+  <tr>
+    <td>Phe/Chl
+    </td>
+    <td>700μL</td>
+  </tr>
+  <tr>
+    <td>Isopropyl alcohol
+    </td>
+    <td>750μL</td>
+  </tr>
+  <tr>
+    <td>75% Ethanol
+    </td>
+    <td>1000μL</td>
+  </tr>
+  </table>
+</div>
+<!--materials終わり-->
+  <br>
+<!--procedure始まり-->
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Add materials (DNA, H2O, CH3COONa, and Phe/Chl) to sample tube and vortex
+    </li>
+    <li>Centrifuge at 14,500 rpm for 10 minutes at room temperature
+    </li>
+    <li>Transfer the supernatant to a new sample tube</li>
+    <li>Add 750 μL of isopropyl alcohol to the sample tube and vortex
+    </li>
+    <li>Centrifuge at 14,500 rpm for 10 minutes at 4°C
+    </li>
+    <li>Remove isopropyl alcohol gently so that the pellet will not break
+    </li>
+    <li>Add 1,000 μL of 75% ethanol and centrifuge at 14,500 rpm for 5 minutes at 4°C
+    </li>
+    <li>Dry it by aspirator for 10 minutes
+    </li>
+    <li>Add 25 μL of distilled H2O and mix
+    </li>
+  </ul>
+<!--procedure終わり-->
+  <br>
+<!--note始まり-->
+  <strong>Note</strong>
+  <ul class="note">
+    <li>Phe/Chl composition     Phenol:Chloroform:Isoamyl alcohol=25:24:1</li>
+  </ul>
+<!--note終わり-->
+  <br>
+  </p>
+<!--本文終わり-->
+  </div>
+  </div>
+<!--DNA refinement 1プロトコル終わり-->
+<!-- DNA Refinement 2プロトコル -->
+  <div class="scroll">
+  </div>
+ <div class="accordion">
+<!--タイトル-->
+  <h2><a href="javascript:void(0);">DNA Refinement 2</a></h2>
+<!--タイトル終わり-->
+  <div class="ac">
+<!--本文-->
+  <p class="sentence">
+<!--materials始まり-->
+  <strong>Materials</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>sample
+    </td>
+    <td>30μL</td>
+  </tr>
+  <tr>
+    <td>Bacterial Alkaline Phosphatase (BAP)
+    </td>
+    <td>1μL</td>
+  </tr>
+  <tr>
+    <td>Low melting agarose gel</td>
+    <td></td>
+  </tr>
+  <tr>
+    <td>6×Loading Buffer Double Dye
+    </td>
+    <td>5μL</td>
+  </tr>
+  <tr>
+    <td>Distilled water
+    </td>
+    <td>200μL</td>
+  </tr>
+  <tr>
+    <td>Phenol
+    </td>
+    <td></td>
+  </tr>
+  <tr>
+    <td>Phe/Chl
+    </td>
+    <td></td>
+  </tr>
+  <tr>
+    <td>Isopropyl alcohol
+    </td>
+    <td>750μL</td>
+  </tr>
+  <tr>
+    <td>5M CH3COONa
+    </td>
+    <td>50μL</td>
+  </tr>
+  <tr>
+    <td>70% Ethanol
+    </td>
+    <td>1000μL</td>
+  </tr>
+  </table>
+</div>
+<!--materials終わり-->
+  <br>
+<!--procedure始まり-->
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Add BAP to vector and dephosphorize for 30 minutes at 37°C
+    </li>
+    <li>Run the electrophoresis with agarose gel
+    </li>
+    <li>Cut out the targeted band and mix with 200 μL of distilled water in a tube</li>
+    <li>Heat the sample for 5 minutes at 65°C
+    </li>
+    <li>Add the same amount of phenol as the sample and vortex
+    </li>
+    <li>Centrifuge at 14,500 rpm for 10 minutes at room temperature and apply the supernatant to the new sample tube
+    </li>
+    <li>Add the same amount of Phe/Chl as the sample and vortex
+    </li>
+    <li>Centrifuge at 14,500 rpm for 5 minutes at room temperature and apply the supernatant to the sample tube
+    </li>
+    <li>Add 750 μL of Isopropyl alcohol and 50 μL of CH3COONa and centrifuge at 14,500rpm for 10 minutes at 4°C
+    </li>
+    <li>Rinse with 70% ethanol at 14,500 rpm 5 minutes at 4°C</li>
+    <li>Dry it for 15 minutes</li>
+  </ul>
+<!--procedure終わり-->
+  <br>
+  </p>
+<!--本文終わり-->
+  </div>
+  </div>
+<!--DNA refinement 2プロトコル終わり-->
 <!--Ligation-->
    <div class="accordion">
@@ Line 302: / Line 839: @@
    <table class="materials">
     <tr>
-     <td colspan="2">DNA sample (cut out from the gel)
+     <td>DNA sample
-    </td>
-   </tr>
-   <tr>
-    <td>Distilled water
      </td>
      <td>5μL</td>
     </tr>
     <tr>
-     <td>DNA ligase
+     <td>DNA ligase (Ligation High from Nippon gene)
      </td>
      <td>5μL</td>
@@ Line 319: / Line 852: @@
    <strong>Procedure</strong>
    <ul class="procedure">
-     <li>Add DNA sample, distilled water and DNA ligase into a micro test tube and vortex
+     <li>Mix DNA sample and DNA ligase in a sample tube gently
      </li>
-     <li>Ligation for 5minutes at room temperature
+     <li>Ligation for 15 minutes at room temperature
      </li>
    </ul>
@@ Line 329: / Line 862: @@
 </div>
 <!--Ligation終わり-->
 <!--Transformation-->
    <div class="accordion">
    <div class="scroll">
    </div>
-   <h2><a href="javascript:void(0);">Transformation (<em>E.coli</em>)</a></h2>
+   <h2><a href="javascript:void(0);">Transformation (<em>E. coli</em>)</a></h2>
    <div class="ac">
    <p class="sentence">
@@ Line 374: / Line 908: @@
 </div>
 <!--Transformation終わり-->
-<!--Pre-culture-->
+<!--Transformation-->
    <div class="accordion">
    <div class="scroll">
    </div>
-   <h2><a href="javascript:void(0);">Pre-culture</a></h2>
+   <h2><a href="javascript:void(0);">Transformation (<em>S. cerevisiae</em>)</a></h2>
    <div class="ac">
    <p class="sentence">
@@ Line 386: / Line 921: @@
      <td>Sample
      </td>
-     <td></td>
+     <td>5μL</td>
     </tr>
     <tr>
-     <td>Medium
+     <td>Competent cells
      </td>
-     <td>20ml</td>
+     <td> 95μL</td>
-    </tr>
+    </tr>
+    <tr>
+    <td>YPD plate containing G418
+    </td>
+    <td></td>
+    </tr>
    </table>
    <br>
    <strong>Procedure</strong>
    <ul class="procedure">
-     <li>Scrape samples from the agar plate and inoculate them on medium
+     <li>Cool down a cuvette for 5 minutes on ice
      </li>
-     <li>Cultivate at 37°C in a shaken culture overnight
+     <li>Harvest the yeast cells by centrifuge at 4 °C
+    </li>
+    <li>Suspend the cells in 20 ml of cold 1M sorbitol and harvest by centrifuge at 4 °C (2times)
+    </li>
+    <li>Suspend the cells in 500μL of cold 1M sorbitol
+    </li>
+    <li>Mix DNA sample (5 μL) and the cell suspension (95 μL) in a cold cuvette
+    </li>
+    <li>Electroporate under the following conditions: 200 Ω, 25 μF and 1.5 kV
+    </li>
+    <li>Immediately inoculate on YPD plates containing G418
+    </li>
+    <li>Incubate at 30 °C for 2 days
      </li>
    </ul>
@@ Line 406: / Line 958: @@
 </div>
 </div>
-<!--Pre-culture終わり-->
+<!--Transformation終わり-->
-<!-- LB Brothプロトコル -->
+<!--Preculture-->
+  <div class="accordion">
    <div class="scroll">
    </div>
- <div class="accordion">
+   <h2><a href="javascript:void(0);">Preculture</a></h2>
-<!--タイトル-->
-   <h2><a href="javascript:void(0);">LB Broth/ LB Medium</a></h2>
-<!--タイトル終わり-->
    <div class="ac">
-<!--本文-->
    <p class="sentence">
-<!--materials始まり-->
    <strong>Materials</strong>
+<div class="m_table">
    <table class="materials">
-  <tr>
+   <tr>
-     <td>Tryptone
+     <td>Sample (<em>E. coli</em> cells )
      </td>
-     <td>final concentration: 1%(w/v)</td>
+     <td></td>
-  </tr>
+   </tr>
-  <tr>
+   <tr>
-     <td>Yeast Extract
+     <td>Medium (LB medium with appropriate antibiotics)
      </td>
-     <td>final concentration: 0.5%(w/v)</td>
+     <td>20ml</td>
-  </tr>
+   </tr>
-  <tr>
-    <td>NaCl</td>
-    <td>final concentration: 1%(w/v)</td>
-  </tr>
-  <tr>
-    <td colspan="2">5M NaOH
-    </td>
-  </tr>
    </table>
-<!--materials終わり-->
+</div>
    <br>
-<!--procedure始まり-->
    <strong>Procedure</strong>
    <ul class="procedure">
-     <li>Dissolve Tryptone (1.0 g), Yeast Extract (500 mg) and NaCl (1.0 g) in distilled water (90 ml)
+     <li>Scrape samples from the agar plate and inoculate them in liquid media
      </li>
-     <li>Adjust pH to 7.0 by adding 20μL of 5M NaOH
+     <li>Cultivate overnight (37℃, 120 rpm)
-    </li>
-    <li>Fill up to 100 ml with distilled water</li>
-    <li>Autoclave
      </li>
    </ul>
-<!--procedure終わり-->
-  <br>
-<!--note始まり-->
-  <strong>Note</strong>
-  <ul class="note">
-    <li>To make agar plate, add agar powder at the final concentration of 2 %(w/v) in Procedure 2</li>
-  </ul>
-<!--note終わり-->
    <br>
    </p>
-<!--本文終わり-->
+</div>
-  </div>
+</div>
-  </div>
+<!--Preculture終わり-->
-<!--プロトコル終わり-->
-<!--YPD Broth-->
+<!--Main Culture-->
-  <div class="accordion">
    <div class="scroll">
    </div>
-   <h2><a href="javascript:void(0);">YPD Broth/YPD Medium</a></h2>
+  <div class="accordion">
+   <h2><a href="javascript:void(0);">Main Culture</a></h2>
    <div class="ac">
    <p class="sentence">
    <strong>Materials</strong>
+<div class="m_table">
    <table class="materials">
     <tr>
-     <td>Peptone
+     <td>LB medium
      </td>
-     <td>final concentration: 2%(w/v)</td>
+     <td>100ml</td>
     </tr>
     <tr>
-    <td>Yeast extract
+  <td>IPTG</td>
-    </td>
+  <td>10μL</td>
-    <td>final concentration: 1%(w/v)</td>
     </tr>
     <tr>
-     <td>Glucose
+     <td>Sample
      </td>
-     <td>final concentration: 2%(w/v)</td>
+     <td>500μL</td>
     </tr>
    </table>
+</div>
    <br>
    <strong>Procedure</strong>
    <ul class="procedure">
-     <li>Dissolve peptone (2.0 g), yeast extract (1.0 g) and glucose (2.0 g) to distilled water (90 ml)
+     <li>Add samples to LB medium and cultivate (37°C, 120 rpm) until reaching a cell density of OD600=0.5</li>
-    </li>
+     <li>Add IPTG (10 μL) to the medium (25 ml)</li>
-     <li>Dilute solution with distilled water and bring up volume to 100 ml
+     <li>Further cultivation for 3 hours
-    </li>
-     <li>Sterilize by autoclave
-    </li>
-  </ul>
-  <br>
-  <strong>Note</strong>
-  <ul class="note">
-    <li>To make agar plate, add agar powder (final concentration: 1.5%) at procedure 1.
      </li>
    </ul>
@@ Line 513: / Line 1,034: @@
 </div>
 </div>
-<!--YPD Broth終わり-->
+<!--Main Culture終わり-->
-<!--Main Culture-->
+<!--Protein Extraction-->
+  <div class="accordion">
    <div class="scroll">
    </div>
-  <div class="accordion">
+   <h2><a href="javascript:void(0);">Protein Extraction (<em>E.  coli</em>)</a></h2>
-   <h2><a href="javascript:void(0);">Main Culture</a></h2>
    <div class="ac">
    <p class="sentence">
    <strong>Materials</strong>
+<div class="m_table">
    <table class="materials">
     <tr>
-     <td>LB medium
+     <td>Sample
      </td>
-     <td>100ml</td>
+     <td></td>
     </tr>
     <tr>
-     <td>IPTG
+     <td colspan="2">FastBreak Cell Lysis Reagent, 10X (Promega)
      </td>
-    <td>10μL</td>
     </tr>
     <tr>
-     <td>Sample
+     <td colspan="2">50mM potassium phosphate buffer (=pH6.8)
+    </td>
+   </tr>
+   <tr>
+    <td colspan="2">SDS sample buffer
      </td>
-    <td>500μL</td>
     </tr>
    </table>
+</div>
    <br>
    <strong>Procedure</strong>
    <ul class="procedure">
-     <li>Add samples to LB medium and cultivate (37℃, 120 rpm) until reaching a cell density of OD600 = 0.5</li>
+     <li>Separate samples into two and harvest by centrifuge
-     <li>Add IPTG (10 μL) to the medium (25 ml)
+    </li>
+    <li>Add potassium phosphate buffer, then mix and remove medium completely
+    </li>
+     <li>Add FastBreak Cell Lysis Reagent, 10X at the ratio of FastBreak Buffer Cell Lysis Reagent,10X: Samples=1:9 and extract protein for 15 minutes at room temperature
      </li>
-    <li>Further cultivation for 3 hours</li>
    </ul>
-  <br>
    <br>
    </p>
 </div>
 </div>
-<!--Main Culture終わり-->
+<!--Protein Extraction終わり-->
 <!--Protein Extraction-->
@@ Line 560: / Line 1,085: @@
    <div class="scroll">
    </div>
-   <h2><a href="javascript:void(0);">Protein Extraction (E.coli)</a></h2>
+   <h2><a href="javascript:void(0);">Protein Extraction (<em>S. cerevisiae</em>)</a></h2>
    <div class="ac">
    <p class="sentence">
    <strong>Materials</strong>
+<div class="m_table">
    <table class="materials">
     <tr>
-     <td>Sample
+     <td>Glass Beads
      </td>
      <td></td>
     </tr>
     <tr>
-     <td colspan="2">Fast Break Cell Lysis Reagent, 10X (Promega)
+     <td colspan="2">50 mM potassium phosphate buffer (pH6.8)
-    </td>
-   </tr>
-   <tr>
-    <td colspan="2">50mM potassium phosphate buffer (=pH6.8)
      </td>
     </tr>
@@ Line 583: / Line 1,105: @@
     </tr>
    </table>
+</div>
    <br>
    <strong>Procedure</strong>
    <ul class="procedure">
-     <li>Separate samples into two and harvest by centrifuge
+     <li>Harvest yeast cells by centrifuge at 3,000 rpm for 4 minutes at room temperature
+    </li>
+<li>Wash cells once with potassium phosphate buffer
      </li>
-     <li>Add potassium phosphate buffer, then mix and remove medium completely
+    <li>Cool down the sample tube on ice for 1 minute
+    </li>
+     <li>Add the same amount of glass beads as the sample then add 20 μL of potassium phosphate buffer and mix
+    </li>
+    <li>Disrupt the cells by FastPrep 24 (MP Biochemicals) for 45 seconds at speed 4.5
+    </li>
+    <li>Cool down the sample tube on ice for 1 minute
+    </li>
+    <li>Add 80 µl of potassium phosphate buffer, cool it down and centrifuge for 5 minutes at 14,500 rpm at 4°C
+    </li>
+    <li>Transfer 40 µl of the supernatant and mix with 8 µl of SDS sample buffer
      </li>
-     <li>Add Fast Break Cell Lysis Reagent, 10X at the ratio of Fast Break Buffer Cell Lysis Readent,10X: Samples=1:9 and extract protein for 15 minutes at room temperature
+     <li>Treat the sample with heat for 5 minutes at 100˚C
      </li>
    </ul>
@@ Line 607: / Line 1,142: @@
    <p class="sentence">
    <strong>Materials</strong>
+<div class="m_table">
    <table class="materials">
     <tr>
@@ Line 621: / Line 1,157: @@
     </tr>
    </table>
+</div>
    <br>
    <strong>Procedure</strong>
@@ Line 646: / Line 1,183: @@
 <!--Colony Sweep終わり-->
-<!--Westone Brotting-->
+<!-- SDS-PAGEプロトコル -->
+  <div class="scroll">
+  </div>
+ <div class="accordion">
+<!--タイトル-->
+  <h2><a href="javascript:void(0);">SDS-PAGE</a></h2>
+<!--タイトル終わり-->
+  <div class="ac">
+<!--本文-->
+  <p class="sentence">
+<!--materials始まり-->
+  <strong>Materials</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>Sample
+    </td>
+    <td></td>
+  </tr>
+  <tr>
+    <td>Separating Gel
+    </td>
+    <td></td>
+  </tr>
+  <tr>
+    <td>Stacking Gel</td>
+    <td></td>
+  </tr>
+    </table>
+</div>
+<!--materials終わり-->
+  <br>
+<!--procedure始まり-->
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Place stacking gel on separating gel
+    </li>
+    <li>Rinse the wells of gel with distilled water
+    </li>
+    <li>Load prepared samples into wells
+    </li>
+    <li>Run the electrophoresis (25 mA for 75 min)
+    </li>
+    <li>Check the bands with CBB stain
+    </li>
+  </ul>
+<!--procedure終わり-->
+  <br>
+<!--Separating Gel始まり-->
+  <strong>Separating Gel</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>Acylamide (%)
+    </td>
+    <td>7.5</td>
+    <td>10</td>
+    <td>12.5</td>
+    <td>15</td>
+    <td>17.5</td>
+  </tr>
+  <tr>
+    <td>MilliQ H2O (ml)
+    </td>
+    <td>3.89</td>
+    <td>3.22</td>
+    <td>2.55</td>
+    <td>1.87</td>
+    <td>1.2</td>
+  </tr>
+  <tr>
+    <td>Acrylamide/Bis-acrylamide (30%/0.8% w/v)</td>
+    <td>1.99</td>
+    <td>2.7</td>
+    <td>3.38</td>
+    <td>4.04</td>
+    <td>4.7</td>
+  </tr>
+  <tr>
+    <td>1.5M Tris-HCl(pH8.8) (ml)</td>
+    <td>2</td>
+    <td>2</td>
+    <td>2</td>
+    <td>2</td>
+    <td>2</td>
+  </tr>
+  <tr>
+    <td>10% (w/v)SDS (μL) </td>
+    <td>80</td>
+    <td>80</td>
+    <td>80</td>
+    <td>80</td>
+    <td>80</td>
+  </tr>
+  <tr>
+    <td>10% (w/v) ammonium persulfate (AP) (μL)</td>
+    <td>27</td>
+    <td>27</td>
+    <td>27</td>
+    <td>27</td>
+    <td>27</td>
+  </tr>
+  <tr>
+    <td>TEMED (μL)</td>
+    <td>4</td>
+    <td>4</td>
+    <td>4</td>
+    <td>4</td>
+    <td>4</td>
+  </tr>
+    </table>
+</div>
+<!--Separating Gel終わり-->
+  <br>
+ <!--Stacking Gel始まり-->
+  <strong>Stacking Gel</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>MilliQ H2O (ml)
+    </td>
+    <td>2.89</td>
+  </tr>
+  <tr>
+    <td>30% (w/v) acrylamide (ml)</td>
+    <td>0.79</td>
+  </tr>
+  <tr>
+    <td>0.5M Tris-HCl(pH6.8) (ml)</td>
+    <td>1.25</td>
+  </tr>
+  <tr>
+    <td>10% SDS (μL) (ml)</td>
+    <td>50</td>
+  </tr>
+<tr>
+    <td>10% APS (μL) (ml)</td>
+    <td>17</td>
+  </tr>
+<tr>
+    <td>TEMED (μl) (ml)</td>
+    <td>5</td>
+  </tr>
+    </table>
+</div>
+<!--Stacking Gel終わり-->
+  <br>
+  </p>
+<!--本文終わり-->
+  </div>
+  </div>
+<!--SDS-PAGEプロトコル終わり-->
+<!--Western Brotting-->
    <div class="accordion">
    <div class="scroll">
@@ Line 654: / Line 1,344: @@
    <p class="sentence">
    <strong>Materials</strong>
+<div class="m_table">
    <table class="materials">
     <tr>
      <td>BufferⅠ
      </td>
-     <td></td>
+     <td> 0.3 M Tris base, 20% MetOH (pH 10.9)</td>
     </tr>
     <tr>
      <td>BufferⅡ
      </td>
-     <td></td>
+     <td>25 mM Tris base, 20% MetOH</td>
     </tr>
     <tr>
      <td>BufferⅢ
      </td>
-     <td></td>
+     <td>25 mM Tris base, 0.05 mM Boric acid, 20% MetOH (pH 9.5)</td>
-   </tr>
-   <tr>
-    <td>Distilled water
-    </td>
-    <td>2ml</td>
     </tr>
     <tr>
      <td>PBS
      </td>
-     <td></td>
+     <td>137 mM NaCl, 8.1 mM Na2HPO4 (12H2O), 2.68 mM KCl, 1.47 mM KH2PO4</td>
     </tr>
     <tr>
      <td>PBS-S
      </td>
-     <td></td>
+     <td>PBS including 0.1% skim milk</td>
     </tr>
     <tr>
      <td>PBS-T
      </td>
-     <td></td>
+     <td>PBS including 0.05% Tween20</td>
     </tr>
     <tr>
      <td>PBS-TS
      </td>
-     <td></td>
+     <td>PBS including 0.1% skim milk & 0.05% Tween20</td>
     </tr>
     <tr>
-     <td>PonceauS
+     <td>PonceauS (Sigma) </td>
-    </td>
      <td></td>
     </tr>
     <tr>
-     <td>PVDF membrane
+     <td>PVDF membrane (Immobilon Transfer membrane by Millipore)
      </td>
      <td>1 sheet</td>
     </tr>
     <tr>
-     <td>Whatman paper
+     <td>Whatman paper (Whatman 3MM)
      </td>
-     <td>6 sheets</td>
+     <td></td>
     </tr>
     <tr>
-     <td>Hybridization bag
+     <td>Hybridization bag (Cosmo Bio)
      </td>
-     <td>1</td>
+     <td></td>
     </tr>
     <tr>
-     <td>Peroxidase Stain Kit
+     <td>Peroxidase Stain Kit (Nacalai Tesque)
      </td>
-     <td>one drop for each</td>
+     <td></td>
     </tr>
     <tr>
-     <td>antiglutathione S - transferase (和光純薬工業株式会社製)
+     <td>Anti-GST antibody conjugated with HRP (Wako)
      </td>
-     <td>1μL</td>
+     <td></td>
     </tr>
    </table>
+</div>
    <br>
    <strong>Procedure</strong>
@@ Line 731: / Line 1,418: @@
      <li>Cut the gel in appropriate size
      </li>
-     <li>Add BufferⅢ and gel then shake it gently
+     <li>Soak gel in buffer III and shake gently for 15 minutes
      </li>
-     <li>Soak the membrane on ethanol then soak it in BufferⅢ and percolate
+     <li>Soak the membrane on methanol then transfer it in buffer III
      </li>
-     <li>2, 1, 3 Whatman papers (all in the same size) on BufferⅠ, BufferⅡ, BufferⅢ respectively. wet the surface of the blotter
+     <li>Soak Whatman papers (all in the same size) in Buffer I (2 sheets), Buffer II (1 sheet) and Buffer III (3 sheets) respectively
      </li>
-     <li>Blot at the constant current of membrane's area ×2.5 mA</li>
+     <li>Wet the surface of the blotter</li>
-<li>Dye the membrane with Ponceau S for 5 minutes rinse it with distilled water and scan it</li>
+<li>Put items in the following order: Buffer III paper, gel, membrane, Buffer II paper and Buffer I paper</li>
-<li>Shake and wash with PBS-TS (3 minutes ×3 times)</li>
+<li>Blot at the constant current of membrane's area ×2.5 mA for 20 minutes</li>
-<li>Put the membrane in a hybridization bag add PBS-S antiglutathione S-transferase and shake it for one hour at room temperature </li>
+<li>Dye the membrane with Ponceau S for 5 minutes, rinse it with distilled water and scan it</li>
-<li>Shake and wash with PBS-T twice (5 min/10 min)</li>
+<li>Shake and wash with PBS-TS (3 minutes ×3times)</li>
-<li>Shake and wash with PBS twice (5 min/5 min)</li>
+<li>The membrane was incubated with anti-GST antibody containing HRP in PBS-S at room temperature for 1 hour</li>
-<li>Add one drip of 3 Peroxidase Stain Kits and distilled water</li>
+<li>Wash with PBS-T twice (5 minutes/10 minutes)</li>
+<li>Wash with PBS twice (5 minutes/5 minutes)</li>
+<li>Stain the membrane using Peroxidase Stain Kits</li>
 <li>Scan it</li>
-</ul>
-<br><strong>Reagent</strong>
-<ul class="materials">
-<li>BufferⅠ: bring to 300 ml with Tris base 10.9g, MetOH60ml/H2O</li>
-<li>BufferⅡ: bring to 300 ml with Tris base 0.9g, MetOH60ml/H2O</li>
-<li>BufferⅢ: bring to 300 ml with Tris base 0.91g, Boric acid10.5mg, MetOH60ml/H2O</li>
-<li>PBS-S: PBS with 1% SkimMilk</li>
-<li>PBS-T: PBS with 0.05% Tween20</li>
-<li>PBS-TS: PBS with 0.05% Tween20+1% SkimMilk</li>
 </ul>
 <br><br></p>
@@ Line 759: / Line 1,439: @@
 </div>
 <!--Westone Botting終わり-->
+<!-- LB Brothプロトコル -->
+  <div class="scroll">
+  </div>
+ <div class="accordion">
+<!--タイトル-->
+  <h2><a href="javascript:void(0);">LB Medium</a></h2>
+<!--タイトル終わり-->
+  <div class="ac">
+<!--本文-->
+  <p class="sentence">
+<!--materials始まり-->
+  <strong>Materials</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>Tryptone
+    </td>
+    <td>final concentration: 1%(w/v)</td>
+  </tr>
+  <tr>
+    <td>Yeast Extract
+    </td>
+    <td>final concentration: 0.5%(w/v)</td>
+  </tr>
+  <tr>
+    <td>NaCl</td>
+    <td>final concentration: 1%(w/v)</td>
+  </tr>
+  <tr>
+    <td colspan="2">5M NaOH
+    </td>
+  </tr>
+  </table>
+</div>
+<!--materials終わり-->
+  <br>
+<!--procedure始まり-->
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Dissolve Tryptone (1.0 g), Yeast Extract (500 mg) and NaCl (1.0 g) in distilled water (90 ml)
+    </li>
+    <li>Adjust pH to 7.0 by adding 20 μL of 5M NaOH
+    </li>
+    <li>Fill up to 100 ml with distilled water</li>
+    <li>Autoclave
+    </li>
+  </ul>
+<!--procedure終わり-->
+  <br>
+<!--note始まり-->
+  <strong>Note</strong>
+  <ul class="note">
+    <li>To make agar plate, add agar powder at the final concentration of 2 %(w/v) in Procedure 2</li>
+  </ul>
+<!--note終わり-->
+  <br>
+  </p>
+<!--本文終わり-->
+  </div>
+  </div>
+<!--LB Mediumプロトコル終わり-->
+<!--YPD Broth-->
+  <div class="accordion">
+  <div class="scroll">
+  </div>
+  <h2><a href="javascript:void(0);">YPD Medium</a></h2>
+  <div class="ac">
+  <p class="sentence">
+<div class="m_table">
+  <strong>Materials</strong>
+  <table class="materials">
+   <tr>
+    <td>Peptone
+    </td>
+    <td>final concentration: 2%(w/v)</td>
+   </tr>
+   <tr>
+    <td>Yeast extract
+    </td>
+    <td>final concentration: 1%(w/v)</td>
+   </tr>
+   <tr>
+    <td>Glucose
+    </td>
+    <td>final concentration: 2%(w/v)</td>
+   </tr>
+  </table>
+</div>
+  <br>
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Dissolve peptone (2.0 g), yeast extract (1.0 g) and glucose (2.0 g) to distilled water (90 ml)
+    </li>
+    <li>Fill up to 100 ml with distilled water
+    </li>
+    <li>Sterilize by autoclave
+    </li>
+  </ul>
+  <br>
+  <strong>Note</strong>
+  <ul class="note">
+    <li>To make agar plate, add agar powder (final concentration: 2.0%) at procedure 1.
+    </li>
+  </ul>
+  <br>
+  </p>
+</div>
+</div>
+<!--YPD Broth終わり-->
+<!-- SD Brothプロトコル -->
+  <div class="scroll">
+  </div>
+ <div class="accordion">
+<!--タイトル-->
+  <h2><a href="javascript:void(0);">SD Medium</a></h2>
+<!--タイトル終わり-->
+  <div class="ac">
+<!--本文-->
+  <p class="sentence">
+<!--materials始まり-->
+  <strong>Materials</strong>
+<div class="m_table">
+  <table class="materials">
+  <tr>
+    <td>Yeast nitrogen base
+    </td>
+    <td>0.67%(w/v)</td>
+  </tr>
+  <tr>
+    <td>Glucose
+    </td>
+    <td>2% (w/v)</td>
+  </tr>
+  <tr>
+    <td>Adenine</td>
+    <td>0.004% (w/v)</td>
+  </tr>
+  <tr>
+    <td>Histidine
+    </td>
+    <td>0.002% (w/v)</td>
+  </tr>
+  <tr>
+    <td>Leucine
+    </td>
+    <td>0.006% (w/v)</td>
+  </tr>
+  <tr>
+    <td>Lysine
+    </td>
+    <td>0.003% (w/v)</td>
+  </tr>
+  <tr>
+    <td>Methionine
+    </td>
+    <td>0.002% (w/v)</td>
+  </tr>
+  <tr>
+    <td>Tryptophan
+    </td>
+    <td>0.004% (w/v)</td>
+  </tr>
+  </table>
+</div>
+<!--materials終わり-->
+  <br>
+<!--procedure始まり-->
+  <strong>Procedure</strong>
+  <ul class="procedure">
+    <li>Dissolve yeast nitrogen base (6.7 g), adenine (40 mg), histidine (20 mg), leucine (60 mg), lysine (30mg), tryptophan (40mg) and methionine (20mg) into 40 ml of distilled H2O, and then fill up to 50 ml with H2O.
+    </li>
+    <li>Dissolve glucose (20.0 g) into 40 ml of distilled H2O, and then fill up to 50 ml with H2O.
+    </li>
+    <li>Sterilize each solution by autoclave.</li>
+    <li>Mix solutions after cooling it down.
+    </li>
+  </ul>
+<!--procedure終わり-->
+  <br>
+<!--note始まり-->
+  <strong>Note</strong>
+  <ul class="note">
+    <li> Autoclave glucose and agar plate separately from other materials</li>
+  </ul>
+<!--note終わり-->
+  <br>
+  </p>
+<!--本文終わり-->
+  </div>
+  </div>
+<!--LB Mediumプロトコル終わり-->
+<!--PCR-1-->
+  <div class="accordion">
+  <div class="scroll">
+  </div>
+  <h2><a href="javascript:void(0);">No. 1 Inverse PCR</a></h2>
+  <div class="ac">
+  <p class="sentence">
+     <ul class="procedure">
+     <li>Prepare PCR primers (10 pmol/μL) and template plasmid (50 ng/μL)</li>
+     <li>Prepare the PCR reaction mixture as follows:
+<div class="m_table">
+  <table class="materials">
+   <tr>
+    <td>PCR grade water</td>
+    <td>35μL</td>
+   </tr>
+   <tr>
+    <td>10×Buffer for iPCR</td>
+    <td>5μL</td>
+   </tr>
+   <tr>
+    <td>2 mM dNTPs</td>
+    <td>5μL</td>
+   </tr>
+   <tr>
+    <td>Primer A (10 pmol/μL)</td>
+    <td>1.5μL</td>
+   </tr>
+   <tr>
+    <td>Primer B (10 pmol/μL)</td>
+    <td>1.5μL</td>
+   </tr>
+   <tr>
+    <td>Plasmid Template DNA (50ng/μL)</td>
+    <td>1μL</td>
+   </tr>
+   <tr>
+    <td>KOD-Plus-DNA Polymerase</td>
+    <td>1μL</td>
+   </tr>
+   <tr>
+    <th>Total</th>
+    <th>50μL</th>
+   </tr>
+  </table>
+</div><br>
+    </li>
+    <li>Perform PCR reaction as follows: <strong>PCR Profile for KOD-FX-NEO</strong>
+<div class="m_table">
+  <table class="materials">
+   <tr>
+    <td>94.0°C</td>
+    <td>98.0°C</td>
+    <td>68.0°C</td>
+    <td>4.0°C</td>
+   </tr>
+   <tr>
+    <td>2 minutes</td>
+    <td>10 seconds</td>
+    <td>7 minutes</td>
+    <td>Hold</td>
+   </tr>
+   <tr>
+    <td>1 cycle</td>
+    <td colspan="2" style="background-color:rgba(0,0,0,0.1);">20 cycles</td>
+    <td>1 cycle</td>
+   </tr>
+  </table>
+  </div><br>
+  </li>
+  <li> [Optional] The control reaction is as follows:
+<div class="m_table">
+  <table class="materials">
+   <tr>
+    <td>PCR grade water</td>
+    <td>35μL</td>
+   </tr>
+   <tr>
+    <td>10×Buffer for iPCR</td>
+    <td>5μL</td>
+   </tr>
+   <tr>
+    <td>2 mM dNTPs</td>
+    <td>5μL</td>
+   </tr>
+   <tr>
+    <td>Control Primer #1（10 pmol/μL）</td>
+    <td>1.5μL</td>
+   </tr>
+   <tr>
+    <td>Control Primer #2（10 pmol/μL）</td>
+    <td>1.5μL</td>
+   </tr>
+   <tr>
+    <td>Control Plasmid pAK119M （50 ng/μL）</td>
+    <td>1μL</td>
+   </tr>
+   <tr>
+    <td>KOD -Plus- (1 U/μL) </td>
+    <td>1μL</td>
+   </tr>
+   <tr>
+    <th>Total</th>
+    <th>50μL</th>
+   </tr>
+  </table>
+</div>
+<br><br>
+<div class="m_table">
+  <table class="materials">
+   <tr>
+    <td>94.0°C</td>
+    <td>98.0°C</td>
+    <td>98.0°C</td>
+   </tr>
+   <tr>
+    <td>2 minutes</td>
+    <td>10 seconds</td>
+    <td>5 minutes</td>
+   </tr>
+   <tr>
+    <td>1 cycle</td>
+    <td colspan="2" style="background-color:rgba(0,0,0,0.1);">5 cycles</td>
+   </tr>
+  </table>
+</div>
+  </li>
+  </ul>
+  <br>
+  </p>
+  </div>
+  </div>
+  <div class="accordion">
+  <div class="scroll">
+  </div>
+  <h2><a href="javascript:void(0);">No. 2 <em>Dpn</em>1 digestion of the template plasmid</a></h2>
+  <div class="ac">
+ 　<ul class="procedure">
+　<li>Add 2 μL of <em>Dpn</em>1 restriction enzyme (10 U/μL) to 50 μL PCR reaction from
+step 1.</li>
+  <li>Gently and thoroughly mix each reaction mixture by pipetting</li>
+  <li>Spin down the reaction mixture and incubate at 37°C for 1 hr to digest the
+template plasmid</li>
+  </ul>
+  <p class="sentence">
+（Optional） Gel electrophoresis<br>
+To confirm PCR amplification, we recommend agarose gel electrophoresis analysis.
+However, in the case of 5 or less cycles of amplification, it may be only possible to
+detect a faint band or no band at all. If only a faint or no band is detected, please
+proceed to the next step. <br><br>
+  </p>
+</div>
+</div>
+  <div class="accordion">
+  <div class="scroll">
+  </div>
+  <h2><a href="javascript:void(0);">No. 3 Self-ligation of the PCR Product</a></h2>
+  <div class="ac">
+  <p class="sentence">
+     <ul class="procedure">
+     <li>Prepare the kination/ligation reaction mixture using a fresh tube as follows:<br>
+<div class="m_table">
+  <table class="materials">
+   <tr>
+    <td><em>Dpn</em>1- treated PCR product </td>
+    <td>2μL</td>
+   </tr>
+   <tr>
+    <td>PCR grade water </td>
+    <td>7μL</td>
+   </tr>
+   <tr>
+    <td>Ligation high</td>
+    <td>5μL</td>
+   </tr>
+   <tr>
+    <td>T4 Polynucleotide Kinase </td>
+    <td>1μL</td>
+   </tr>
+   <tr>
+    <th>Total Volum</th>
+    <th>15μL</th>
+   </tr>
+  </table>
+</div><br>
+    </li>
+    <li>
+Gently and thoroughly mix each reaction mixture by pipetting
+  </li>
+  <li>Centrifuge the reaction mixture and incubate each reaction at 16°C for 1 hr
+  </li>
+  </ul>
+  <br>
+  <br>
+  </p>
+  </div>
+  </div>
+  <div class="accordion">
+  <div class="scroll">
+  </div>
+  <h2><a href="javascript:void(0);">No. 4 Transformation</a></h2>
+  <div class="ac">
+  <p class="sentence">
+Standard <em>E. coli</em> strains can be used as the transformation host. Both
+electrocompetent and chemically competent <em>E. coli</em> cells can be used. The
+following protocol is a standard protocol using chemically prepared competent <em>E.
+coli</em> cells. <br>
+  <ul class="procedure">
+  <li>Gently thaw the competent cells （100 μL） on ice. </li>
+  <li>Add 10 μL of reaction mixture from Step 3 to the competent cells, swirl gently
+and incubate for 30 min on ice.</li>
+  <li>Incubate for 30 seconds at 42°C and then place on ice for 2 min</li>
+  <li>Add 900 μL of SOC medium and incubate at 37°C for 1 hr with shaking.
+</li>
+  <li>Plate appropriate volumes (10~200 μL) of transformed <em>E. coli</em> solution on agar
+plates containing the appropriate antibiotic for the plasmid vector. </li>
+  <li>Incubate the plates at 37°C for ~16 hr.</li>
+  </ul>
+<br>
+<br>
+</p>
+</div>
+</div>
+  <div class="accordion">
+  <div class="scroll">
+  </div>
+  <h2><a href="javascript:void(0);">No. 5 Analysis of Transfomants</a></h2>
+  <div class="ac">
+  <p class="sentence">
+A typical mutagenesis efficiency of this kit is greater than 80%. Therefore, analysis
+of 4-8 clones is typically appropriate for sequence verification. <br><br></p></div></div>
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+<style type="text/css">
+ul.slidemenu{
+height:300px;
+overflow:scroll;
+}
+h2 em{
+font-size:30px;
+}
+</style>
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Team:KIT-Kyoto/Notebook/Protocol

From 2014.igem.org

Latest revision as of 03:36, 18 October 2014

Protocol