Team:KIT-Kyoto/c6h12o6

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Protocol

Miniprep

Solution Ⅰ (50 mM Tris-HCl and 10 mM EDTA, and 50 µg/mL RNase A, pH 8.0 (25˚C))

Solution Ⅱ (0.2 M NaOH and 1 % SDS)

Solution Ⅲ (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)

Solution Ⅳ (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH 6.6 (25˚C))

Solution Ⅴ (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25˚C))

Pick up a single colony from plate and cultivate it overnight in 3ml LB medium containing appropriate antibiotic at 37˚C˚.

↓

Transfer the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute, and discard the supernatant (2 times).

↓

Add 250 µL of SolutionⅠ to the pellet and mix well with vortex.

↓

Add 250 µL of SolutionⅡ and mix well by turning the tube upside down several times.

↓

Add 350 µL of SolutionⅢ and by turning the tube upside down several times.

↓

Centrifuge at 13,000 rpm for 5 minutes.

↓

Transfer the supernatant (approx. 850µL) to a mini prep column.

↓

Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.

↓

Add 500 µL of SolutionⅣ.

↓

Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.

↓

Add 700 µL of SolutionⅤ.

↓

Centrifuge at 13,000 rpm for 1 minute and discard the flow-through (2 times).

↓

Set the column on a new 1.5ml tube and add 100 µL of nuclease-free water.

↓

Centrifuge at 13,000 rpm for 1 minute and collect plasmid DNA.

Rapid check of the insert by colony cracking

Add 45 µL of cracking solution(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube.

↓

Suspend a small quantity of the colony in a tube using a sterilized toothpick.

↓

Incubate the tube at 65°C for 10 minutes.

↓

Add a drop of 10x Loading Buffer.

↓

Add equal volume of phenol/chloroform.

↓

Mix with vortex.

↓

Centrifuge at 13,000rpm for 3 minutes.

↓

Apply the supernatant to 1% agarose gel electrophoresis.

↓

Stain the gel in ethidium bromide solution for 10 minutes.

↓

Plasmid DNA can be detected by UV-illuminator as a band between genomic DNA band and low molecular size RNAs.

DNA purification and precipitation

Mix 350 µL of sterilized water and 50 µL of 3 M sodium acetate with DNA sample.
Mix DNA solution with equal volume of phenol/chloroform by vortex.
Centrifuge at 13,000 rpm for 5 min, then transfer the supernatant into a new tube.
Add equal volume of 2-propanol, and mix by turning the tube upside down.
Centrifuge at 14,500 rpm for 10 min at 4˚C.
Carefully decant the supernatant.
Add adequate volume of 70 % ethanol.
Centrifuge at 14,500 rpm for 5 min at 4˚C.
Carefully decant the supernatant.
Dry in the desiccator under vacuum for 5 min.
You can get dried DNA pellet.

PCR

Adjust the concentration of each primer to 100 pmol/µL with sterilized water.
Mix 10µL of forward and reverse primer solutions with 80 µL H₂O in a new tube (final primer concentration is 10 pmol/µL).
Use 1 µL of primer mix for PCR.

Recipe for PRC is as follows:

Buffer	50 µL
dNTP	20 µL
Primer mix	1 µL
DNA sample	0.5 µL
KOD-FX	2 µL
H₂O	26.5 µL
total	100 µL

KOD-FX(Toyobo)

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

12.5% separation gel (recipe for a sheet of gel)

Mili Q water 2.59 ml

acrylamide solution(30 %) 3.33 ml

0.5M Tris (pH8.8) 2 ml

10%SDS 80 µL

10%APS 27 µL

TEMED 4 µL

↓

Apply the acrylamide solution mix to the PAGE glass plate.

↓

Deposit H₂O carefully on the top of the acrylamide solution mix.

↓

Wait for 10 min for polymerization.

Stacking gel

Mili Q water 2.89 ml

Acrylamide 0.79 ml

0.5M Tris (pH 6.8) 1.25 ml

10% SDS 50 µL

10%APS 17 µL

TEMED 5 µL

↓

After the polymerization of the separation gel, remove the H₂O of the top layer.

↓

Apply stacking gel mix on the running gel and put the comb to make wells.

↓

After the stacking gel has fully polymerized, remove the comb and rinse the top of the gel with H₂O and then remove H₂O.

Sample preparation

Collect bacterial cells.

↓

Add 450ul of H₂O and 50 µL of FastBreak Cell Lysis Reagent (Madison, Wisconsin, USA Promega).

↓

Mix them for 15minutes with shaker.

↓

Add 100ul of 6ｘSDS-Sample buffer and mix with vortex.

↓

Heat samples in a heating block at 99°C for 5 minutes.

Running samples

Set the gel on the electrophoresis apparatus and apply SDS running buffer (Tris 25mM,Glysine 191mM,SDS0.1%)

↓

Apply the samples and markers.

↓

Operate at constant current (25mA) for 65 minutes per sheet of gel.

Staining

Place the gel in stacking solution( 50％ethanol,10％acetic acid) and shake gently it for 5 minutes.

↓

Remove the stacking solution and add 100ml of staining solution (0.25% CBB R250、5%methanol、7.5%acetic acid).

Warm the gel for 1 minute with a microwave (500W).

↓

Remove the staining solution and rinse the gel with water.

Isolation and purification of DNA bands

Clip DNA band from agarose gel and transfer it into a 1.5 ml tube.
Add H₂O. Melt the gel at 65˚C in a heating block.
Add equal volume of phenol. Mix well with vortex.
Centrifuge at 13,000 rpm for 5 min.
Transfer the supernatant into a new tube and mix with equal volume of phenol/chloroform.
Perform the same method of DNA precipitation using 2-propanol.

Blue gel
・50×TAE 100ml
・Agarose(nacalai tesque) 1.0g
・Gel indicator(Biodynamics laboratory) 200ul

<Ligation>

・Dissolve the purified and dried insert DNA in 5µL of H₂O.

・Dissolve the purified and dried vector DNA in 5µL of H2O.

・Mix the two solutions.

・Add 10µL of Ligation Mix (Wako) to it.

・Incubate for 15 minutes at room temperature.

<Transformation>

・Add the ligation solution to competent cells (in a clean bench).

・Place tubes on ice for 20 minutes.

・Heat shock treatment at 42°C for 35 seconds.

・Place tubes on ice for 2 minutes.

・Add 1mL of LB medium into the tube (in a clean bench).

・Incubate the samples for 20 minutes at 37°C.

・Centrifuge at 7,000rpm for 3 minutes.

・Discard the most of supernatant, and spread the remaining cells and LD medium in the tube on the plates (in a clean bench).

・Incubate plates overnight at 37°C.

August 1st

Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.

① We performed PCR in the following conditions.

(ⅰ)TNFAIP3
Composition for reaction

	sample1
10ng/μL TNFAIP3	6μL
10× KOD plus buffer	10μL
2mM dNTPs	10μL
25mM MgSO₄	3.2μL
10P 5'primer	3μL
10P 3'primer	3μL
KOD plus	2μL
dH₂O	62.8μL
Total	100μL

Reaction conditions

temperature	time	cycle
95°C	2min.
95°C	15sec	25cycle
60°C	30sec	25cycle
68°C	2min30sec	25cycle
68°C	2min30sec
14°C	∞

August 2nd

① PCR product check

We run 0.7% agarose gel electorophoresis in the following conditions. Composition

	8/1 the attB TNFAIP3 PCR products
DNA sample	10μL
6×Dye	2μL
total	12μL

The order of sample application
Left : 1kb marker(5uL)
Right: attB TNFAIP3

Results

②　DNA　purification from gel
We electrophoresed in 0.7% agarose gel in the following conditions.

Composition

	8/1 attB TNFAIP3 PCRproduct
DNA sample	90μL
6×Dye	18μL
total	108μL

We applied half of 108 uL of sample to each of two lanes of Agaroe gel with 5uL of 1kb marker.Then we detected the DNA band in the gel.

Results

We purifed DNA from the gel by QIA Quick Gel Extraction Kit.

・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.
Order of samples applied to the agarose gel
The samples were electrophoresed in 0.7% agarose gel in following order.
Right to left:3uL of 1kb marker 1uL of 5 fold dilution of attB TNFAIP3 DNA fragments,1uL of 10 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 1kb marker.

Results

We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.

August 3rd

①BP reaction

BP reaction was carried out in the following conditions.

attB TNFAIP3(80ng/μL)	1μL
pDONR(455ng/μL)	0.35μL
TE buffer	7.65
total	9μL

One uL of BP Clonase Ⅱ enzyme mix was added to this solution and incubated for 2.5 hours.

② Transformation of E.coli by BP reaction products
2uL of BP TNFA reaction product was added to 100uL of E.coli XL1-Blue to do transform ation.
At last, cells were divided into 10,40,100,and 200 uL,and spreaded on LB Kanamycin(+)plate,Plats wera incubated for 16 hours at 37°C.

August 4th

Four independent 4 colonies were picked up from the plate incubated from 8/3 as described in ②,then cultured in 2.5mL of LB Kanamycin(+) liquid culture medium for 16 hours.

August 5th

Plasmid DNAs were isolated from E.coli cultured in 4 of LB liquid culture medium which we made on 8/4 by the alkaline-lysis method, we added the DNA to 20uL TE and added 10uL of the solution to 2uL of 6×Dye. Then we electrophoresed it with marker, 2uL of pDONR+ 2uL of 6×Dye+ 8uL of TE,in 0.7% agarose gel in following order (from left to right)

Left control pDONR DNA, BP TNFA-1,-2,-3,-4

Results

August 6th

E.coli carrying the plasmid sample -1 and -2 in LB Kanamycin(+)liquid culture medium for 16 hours.

August 7th

①Purification of candidate pENTR-TNFAIP3
We purified candidate plasmid-1 and -2 by QIA Prep Spin Miniprep Kit, dissolved it in 30uL of Buffer EB.
Then,the samples were electrophoresed in 0.7% agarose gel.

We made samples as follows.

	pDONR	BP TNFA-1	BP TNFA-2
DNA sample	1μL	1μL	1μL
6×Dye	1μL	1μL	1μL
dH₂O	4μL	4μL	4μL
total	6μL	6μL	6μL

We electrophoresed them in following order.
Left marker 5uL control pDONR DNA ,pENTR-TNFAIP3-1 DNA,pENTR-TNFAIP3 -2 DNA marker 4uL marker 3uL Wright.

Results

Concentration of pENTR-TNFAIP3-2 DNA was estimated at around 100ng/uL.

②　LR reaction
We made following solution(vials)in 1.5mL tube.

BP TNFA-2	2μL
pTFW(Destination vector)	0.5μL
TE buffer	6.5μL
total	9μL

we added 1uL of LR clonaseⅡ enzyme mix to this solution and incubate it for 2.5 hours.

③ Transformation of E.coli with LR reaction products.
Transformation of E.coli was carried out by adding 2uL of LR reaction products to 100uL of XL1-Blue and plated on the LB ampicillin(+)plate and cultured at 37°C.

WEEK１終わり

August 8th

We isolated 5 colonies from the LB plate, and cultured.

August 9th

①Purification of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.
We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.

②Characterization of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.

Each diluted DNA sample	1μL
10×H buffer	0.5μL
EcoRⅠ	0.2μL
dH₂O	3.3μL
total	5μL

After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.
Left,　10fold dilution,　2uL marker,　20fold dilution,　30fold dilution,　1uL marker,　40fold dilution,　0.5uL marker right.

Results

The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.

③ PCR amplification of the insert DNA

The four 5'primers were designed for PCR as follows.

・GW3r
5’-ATCGAGGCCTGTCTAGAGAAGC
・TNFA-1
5’-GGCTTTGCTATGATACTCGGAACTG
・TNFA-2
5’-GTAAAATGTGAAACGCCCAACTGC
・TNFA-3
5’-GGACTCCAGAAAACAAGGGCTTT

The 3’ primer was designed as follows.
・SVr
5’-GGCATTCCACCACTGCTCCC

PCR reactions with the following combinations of primers were carried out.
sample１→GW3r―SVr
sample２→TNFA-1―SVr
sample３→TNFA-2―SVr
sample４→TNFA-3―SVr

PCR was carried out in the following reactions.

	Each sample
50ng/μL LR TNFA-1	1μL
10×rTaq buffer	2μL
2mM dNTPs	2μL
25mM MgCl₂	0.8μL
10P 5'primer	0.6μL
10P 3'primer	0.6μL
rTaq	0.4μL
dH₂O	12.6μL
total	20μL

Reaction conditions of PCR

temperature	time	cycle
95°C	2min
95°C	15sec	25cycle
55°C	30sec	25cycle
68°C	2min30sec	25cycle
68°C	2min30sec
14°C	∞

August 10th

Electrophoresis of PCR products was carried out.
After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.

Left　marker5uL　sample１　sample２　sample３　sample４　Wright

Results

The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.

August 13th

Cut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction.

The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.

the candidate pENTR-TNFAIP3 prepared on 8/6	1μL
10×M buffer	0.5μL
Hind Ⅲ	0.2μL
dH₂O	3.3μL
total	5μL

After reaction, samples were electrophoresed in following order.

Left: 1kb marker5uL cut sample
Right: uncut sample

Results

Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful.

August 14th

Based on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again.

Composition of the reaction

	sample1
10ng/μL TNFAIP3	6μL
10×KOD plus buffer	10μL
2mM dNTPs	10μL
25mM MgSO₄	3.2μL
10P 5'primer	3μL
10P 3'primer	3μL
KOD plus	2μL
dH₂O	62.8μL
total	100μL

Reaction conditions

temperature	time	cycle
95°C	2min
95°C	15sec	25cycle
60°C	30sec	25cycle
68°C	2min30sec	25cycle
68°C	2min30sec
14°C	∞

PCR product was applied to the 0.7% agarose gel electrophoresis.

Results

WEEK2終わり

August 20th

1. Reproduction of DNA from gel
We did agarose gel electrophoresis (0.7% gel).

Composition

	a product of PCR attB TNFAIP3(8/1)
DNA sample	90μL
6×Dye	18μL
Total	108μL

We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL).We separated gel containing DNA.

Results
We isolated DNA from these gels by QIA quick Gel Extraction Kit.
Finally we melt DNA to TE Buffer(40uL)

August 21st

TNFA and API2

1. Density measurement of attB TNFAIP3
We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )

Results
We estimated attB TNFAIP3(we make this time) is 35ng/uL

2. BP reaction
We adjusted solution (vials) on 1.5mL tube to next composition.

attB TNFAIP3(35ng/μL)	2μL
pDONR(150ng/μL)	1μL
TE buffer	5μL
Total	8μL

We added BP Clonase Ⅱ enzyme mix-2uL to this vials.
We incubated these vials for 2hour at 25˚C. Next we did BP reaction

3. Transformation
We added BP TNFAIP3-2uL to XL1-Blue-100uL and we did Transformation.
Finally we spread 10uL, 40uL and 100uL on plate of LB Kanamycin(＋) and we keep 37℃ overnight.

August 22nd

TNFA and API2

We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .
We cultivated these colonies on 2.5mL Kanamycin(＋) LB culture medium and we kept shaking this medium overnight.

DNA purification and precipitation

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+  <div id="doc-left">
+{{Template:KIT-Kyoto/menu_note}}
+  </div>
+<div id="doc-right" class="metro">
+<h2>Protocol</h2>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 13pt"><B>Miniprep</B></FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+&#8544; (50 mM Tris-HCl and 10 mM EDTA, and 50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">g/mL
+RNase A, pH 8.0 (25&#730;C))</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+&#8545; (0.2 M NaOH and 1 % SDS)</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+&#8546; (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+&#8547; (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH
+.6 (25&#730;C))</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+&#8548; (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25&#730;C))</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt"></FONT></FONT></FONT></P>
+<br>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Pick
+up a single colony from plate and cultivate it overnight in 3ml LB
+medium containing appropriate antibiotic at 37&#730;C&#730;.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Transfer
+the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute,
+and discard the supernatant (2 times).</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+of Solution&#8544; to the pellet and mix well with vortex.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+of Solution&#8545; and mix well by turning the tube upside down several
+times.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+of Solution&#8546; and by turning the tube upside down several times.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+at 13,000 rpm for 5 minutes.</FONT></SPAN></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Transfer
+the supernatant (approx. 850</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L)
+to a mini prep column.</FONT></SPAN></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+at 13,000 rpm for 1 minute and discard the flow-through.</FONT></SPAN></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+of Solution&#8547;.</FONT></SPAN></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+at 13,000 rpm for 1 minute and discard the flow-through.</FONT></SPAN></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+of Solution&#8548;.</FONT></SPAN></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+at 13,000 rpm for 1 minute and discard the flow-through (2 times).</FONT></SPAN></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Set
+the column on a new 1.5ml tube and add 100 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+of nuclease-free water.</FONT></SPAN></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+at 13,000 rpm for 1 minute and collect plasmid DNA.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">	</SPAN></FONT></P>
+<BR>
+<P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 13pt"><B>Rapid
+check of the insert by colony cracking</B></FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+</FONT>&micro;L<FONT SIZE=2 STYLE="font-size: 11pt"> of cracking
+solution(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube.   </FONT></SPAN></FONT></p>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Suspend
+a small quantity of the colony in a tube using a sterilized
+toothpick.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Incubate
+the tube at 65&#176;C for 10 minutes.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+a drop of 10x Loading Buffer.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+equal volume of phenol/chloroform.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mix
+with vortex.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+at 13,000rpm for 3 minutes.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
+the supernatant to 1% agarose gel electrophoresis.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Stain
+the gel in ethidium bromide solution for 10 minutes.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Plasmid
+DNA can be detected by UV-illuminator as a band between genomic DNA
+band and low molecular size RNAs.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<UL>
+	<LI><P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><B>
+	</B></FONT><FONT FACE=""><SPAN LANG="en-US"><B>DNA purification and
+	precipitation</B></SPAN></FONT></P>
+</UL>
+<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
+</P>
+<OL>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
+&#181;L of sterilized water and 50 &#181;L of 3 M sodium
+	acetate with DNA sample.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
+	DNA solution with equal volume of phenol/chloroform by vortex.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
+	at 13,000 rpm for 5 min, then transfer the supernatant into a new
+	tube.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
+	equal volume of 2-propanol, and mix by turning the tube upside down.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
+	at 14,500 rpm for 10 min at 4&#730;C.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Carefully
+	decant the supernatant.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
+	adequate volume of 70 % ethanol.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
+	at 14,500 rpm for 5 min at 4&#730;C.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Carefully
+	decant the supernatant.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Dry
+	in the desiccator under vacuum for 5 min.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">You
+	can get dried DNA pellet.</SPAN></FONT></P>
+</OL>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><FONT COLOR="#000000"></FONT></FONT></P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm; page-break-before: always"><BR>
+</P>
+<UL>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>PCR</B></SPAN></FONT></P>
+</UL>
+<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
+</P>
+<OL>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Adjust
+	the concentration of each primer to 100 pmol/&micro;L with
+	sterilized water.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
+&micro;L of forward and reverse primer solutions with 80 &micro;L
+	H<SUB>2</SUB>O in a new tube (final primer concentration is 10
+	pmol/&micro;L).</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Use
+&micro;L of primer mix for PCR.</SPAN></FONT></P>
+</OL>
+<P STYLE="margin-left: 1.48cm; margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Recipe
+for PRC is as follows:</SPAN></FONT></P>
+<TABLE WIDTH=206 BORDER=1 BORDERCOLOR="#00000a" CELLPADDING=7 CELLSPACING=0>
+	<TR VALIGN=TOP>
+		<TD WIDTH=106>
+			<P><FONT FACE=""><SPAN LANG="en-US">Buffer</SPAN></FONT></P>
+		</TD>
+		<TD WIDTH=70>
+			<P><FONT FACE=""><SPAN LANG="en-US">50 &micro;L</SPAN></FONT></P>
+		</TD>
+	</TR>
+	<TR VALIGN=TOP>
+		<TD WIDTH=106>
+			<P><FONT FACE=""><SPAN LANG="en-US">dNTP</SPAN></FONT></P>
+		</TD>
+		<TD WIDTH=70>
+			<P><FONT FACE=""><SPAN LANG="en-US">20 &micro;L</SPAN></FONT></P>
+		</TD>
+	</TR>
+	<TR VALIGN=TOP>
+		<TD WIDTH=106>
+			<P><FONT FACE=""><SPAN LANG="en-US">Primer mix</SPAN></FONT></P>
+		</TD>
+		<TD WIDTH=70>
+			<P> <FONT FACE=""><SPAN LANG="en-US">1 &micro;L</SPAN></FONT></P>
+		</TD>
+	</TR>
+	<TR VALIGN=TOP>
+		<TD WIDTH=106>
+			<P><FONT FACE=""><SPAN LANG="en-US">DNA sample</SPAN></FONT></P>
+		</TD>
+		<TD WIDTH=70>
+			<P><FONT FACE=""><SPAN LANG="en-US">0.5 &micro;L</SPAN></FONT></P>
+		</TD>
+	</TR>
+	<TR VALIGN=TOP>
+		<TD WIDTH=106>
+			<P><FONT FACE=""><SPAN LANG="en-US">KOD-FX</SPAN></FONT></P>
+		</TD>
+		<TD WIDTH=70>
+			<P> <FONT FACE=""><SPAN LANG="en-US">2 &micro;L</SPAN></FONT></P>
+		</TD>
+	</TR>
+	<TR VALIGN=TOP>
+		<TD WIDTH=106>
+			<P><FONT FACE=""><SPAN LANG="en-US">H<SUB>2</SUB>O</SPAN></FONT></P>
+		</TD>
+		<TD WIDTH=70>
+			<P><FONT FACE=""><SPAN LANG="en-US">26.5 &micro;L</SPAN></FONT></P>
+		</TD>
+	</TR>
+	<TR VALIGN=TOP>
+		<TD WIDTH=106>
+			<P><FONT FACE=""><SPAN LANG="en-US">total</SPAN></FONT></P>
+		</TD>
+		<TD WIDTH=70>
+			<P><FONT FACE=""><SPAN LANG="en-US">100 &micro;L</SPAN></FONT></P>
+		</TD>
+	</TR>
+</TABLE>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">KOD-FX</SPAN></FONT><FONT FACE=""></FONT><FONT FACE=""><SPAN LANG="en-US">(</SPAN></FONT><FONT FACE=""><FONT COLOR="#000000">Toyobo</FONT></FONT><FONT FACE=""><SPAN LANG="en-US">)</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">SDS
+polyacrylamide gel electrophoresis (SDS-PAGE)</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>12.5%
+separation gel (recipe for a sheet of gel)</B></FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mili
+Q water			2.59 ml </FONT></SPAN></FONT>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">acrylamide
+solution(</FONT><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">30
+</FONT></FONT><FONT SIZE=2 STYLE="font-size: 11pt">%)	3.33 ml</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">0.5M
+Tris (pH8.8)		2 ml </FONT></SPAN></FONT>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%SDS			80
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+</FONT></SPAN></FONT>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%APS			27
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+</FONT></SPAN></FONT>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">TEMED			4
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+</FONT></SPAN></FONT>
+</P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
+the acrylamide solution mix to the PAGE glass plate.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Deposit
+H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
+carefully on the top of the acrylamide solution mix.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Wait
+for 10 min for polymerization.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Stacking
+gel  </B></FONT></SPAN></FONT>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mili
+Q water 	2.89 ml</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Acrylamide
+.79 ml</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">0.5M
+Tris (pH 6.8) 	1.25 ml</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%
+SDS 	50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%APS
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">TEMED
+</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">After
+the polymerization of the separation gel, remove the H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
+of the top layer.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
+stacking gel mix on the running gel and put the comb to make wells.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">After
+the stacking gel has fully polymerized, remove the comb and rinse the
+top of the gel with H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
+and then remove H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Sample
+preparation</B></FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Collect
+bacterial cells.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+ul of H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
+and 50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+of FastBreak Cell Lysis Reagent (</FONT><FONT SIZE=2 STYLE="font-size: 11pt">Madison,
+Wisconsin, USA</FONT><FONT SIZE=2 STYLE="font-size: 11pt"> Promega).</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mix
+them for 15minutes with shaker.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+ul of 6</FONT></SPAN></FONT><FONT FACE=""><FONT SIZE=2 STYLE="font-size: 11pt">ｘ</FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">SDS-Sample
+buffer and mix with vortex.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Heat
+samples in a heating block at 99&#176;C for 5 minutes.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Running
+samples</B></FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Set
+the gel on the electrophoresis apparatus and apply SDS running buffer
+(</FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">Tris 25mM,Glysine 191mM,SDS0.1%</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">)
+</FONT></SPAN></FONT>
+</P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
+the samples and markers.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Operate
+at constant current (25mA) for 65 minutes per sheet of gel.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Staining</B></FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Place
+the gel in stacking solution( </FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">50％ethanol,10％acetic acid</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">)
+and shake gently it for 5 minutes.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Remove
+the stacking solution and add 100ml of staining solution (</FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">0.25% CBB R250、5%methanol、7.5%acetic acid</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">).</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Warm
+the gel for 1 minute with a microwave (500W).</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm">&darr;</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Remove
+the staining solution and rinse the gel with water.</FONT></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<UL>
+	<LI><P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><B></B></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>Isolation
+	and purification of DNA bands</B></SPAN></FONT></P>
+</UL>
+<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
+</P>
+<OL>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Clip
+	DNA band from agarose gel and transfer it into a 1.5 ml tube.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
+	H<SUB>2</SUB>O. Melt the gel at 65&#730;C in a heating block.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
+	equal volume of phenol. Mix well with vortex.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
+	at 13,000 rpm for 5 min.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Transfer
+	the supernatant into a new tube and mix with equal volume of
+	phenol/chloroform.</SPAN></FONT></P>
+	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Perform
+	the same method of DNA precipitation using 2-propanol.</SPAN></FONT></P>
+Blue gel<br>
+・50×TAE 100ml<br>
+・Agarose(nacalai tesque) 1.0g<br>
+・Gel indicator(Biodynamics laboratory) 200ul<br>
+</OL>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>&lt;Ligation&gt;</B></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Dissolve
+the purified and dried insert DNA in 5&#181;L of H<SUB>2</SUB>O.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Dissolve
+the purified and dried vector DNA in 5&#181;L of H2O.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Mix
+the two solutions.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
+&micro;L of Ligation Mix (</SPAN></FONT><FONT FACE=""><FONT COLOR="#000000">Wako</FONT></FONT><FONT FACE=""><SPAN LANG="en-US">)
+to it.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
+for 15 minutes at room temperature.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>&lt;Transformation&gt;</B></SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
+the ligation solution to competent cells (in a clean bench).</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Place
+tubes on ice for 20 minutes.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Heat
+shock treatment at 42&#176;C for 35 seconds.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Place
+tubes on ice for 2 minutes.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
+mL of LB medium into the tube (in a clean bench).</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
+the samples for 20 minutes at 37&#176;C.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Centrifuge
+at 7,000rpm for 3 minutes.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Discard
+the most of supernatant, and spread the remaining cells and LD medium
+in the tube on the plates (in a clean bench).</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
+plates overnight at 37&#176;C.</SPAN></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+</div>
+</div>
 {{KIT-Kyoto}}
 <html>
@@ Line 6: / Line 567: @@
 <h2>August 1st</h2>
-<br>
-<strong>*TNFAIP3</strong>
 <br>
 <br>
-Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.
+Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.<br>
-<br>
 <br>
 ① We performed PCR in the following conditions.<br>
 <br>
-<strong>primer</strong><br>
-<br>
 (ⅰ)TNFAIP3<br>
 Composition for reaction
@@ Line 45: / Line 602: @@
 </Table>
 <br>
+<br>
 <h2>August 2nd</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
 ① PCR product check<br>
@@ Line 69: / Line 625: @@
 Left : 1kb marker(5uL)<br>
 Right: attB TNFAIP3<br>
+<br>
 <strong>Results</strong>
@@ Line 97: / Line 654: @@
 We purifed DNA from the gel by QIA Quick Gel Extraction Kit.<br>
-<br>
+<br><br>
 ・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.<br>
-<br>
 Order of samples applied to the agarose gel<br>
 The samples were electrophoresed in 0.7% agarose gel in following order.<br>
-<br>
 Right to left:3uL of 1kb marker 1uL of 5 fold dilution of attB TNFAIP3 DNA fragments,1uL of 10 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 1kb marker.<br>
 <br>
@@ Line 110: / Line 665: @@
 <img src="https://static.igem.org/mediawiki/2012/0/0e/0802-3.png" width="500" height="300">
 <br>
-We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.<br><br>
+We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.<br>
+<br>
 <br>
-<strong>Result</strong>
-<br><br>
 <h2>August 3rd</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
+①BP reaction<br>
+<br>
+BP reaction was carried out in the following conditions.
 <Table Border Cellspacing="0">
 <Tr><Td>　attB TNFAIP3(80ng/μL)　</Td><Td>　1μL　</Td></Tr>
@@ Line 132: / Line 684: @@
 </Table>
 <br>
+<br>
+One uL of BP Clonase Ⅱ enzyme mix was added to this solution and incubated for 2.5 hours.<br>
+<br>
+<br>
+② Transformation of E.coli by BP reaction products<br>
+uL of BP TNFA reaction product was added to 100uL of E.coli XL1-Blue to do transform ation.<br>
+At last, cells were divided into 10,40,100,and 200 uL,and spreaded on LB Kanamycin(+)plate,Plats wera incubated for 16 hours at 37°C.<br>
 <br>
 <br>
@@ Line 138: / Line 696: @@
 <h2>August 4th</h2>
-<br>
-<strong>*TNFA and API2</strong>
 <br><br>
+Four independent 4 colonies were picked up from the plate incubated from 8/3 as described in ②,then cultured in 2.5mL of LB Kanamycin(+) liquid culture medium for 16 hours.<br>
+<br>
+<br>
 <h2>August 5th</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
-<br><br>
-<img src="https://static.igem.org/mediawiki/2012/3/31/0805.png" width="500" height="300">
-<br><br>
-<h2>August 6th</h2>
+Plasmid DNAs were isolated from E.coli cultured in 4 of LB liquid culture medium which we made on 8/4 by the alkaline-lysis method, we added the DNA to 20uL TE and added 10uL of the solution to 2uL of 6×Dye. Then we electrophoresed it with marker, 2uL of pDONR+ 2uL of 6×Dye+ 8uL of TE,in 0.7% agarose gel in following order (from left to right)<br>
+<br>
+Left control pDONR DNA, BP TNFA-1,-2,-3,-4<br>
 <br>
-<strong>*TNFA and API2</strong>
-<br><br>
+<strong>Results</strong><br>
+<img src="https://static.igem.org/mediawiki/2012/3/31/0805.png" width="500" height="300"><br>
+<br>
+<br>
+<h2>August 6th</h2>
+<br>
+<br>
+E.coli carrying the plasmid sample -1 and -2 in LB Kanamycin(+)liquid culture medium for 16 hours.<br>
+<br>
+<br>
 <h2>August 7th</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
+①Purification of candidate pENTR-TNFAIP3<br>
+We purified candidate plasmid-1 and -2 by QIA Prep Spin Miniprep Kit, dissolved  it in 30uL of Buffer EB.<br>
+Then,the samples were electrophoresed in 0.7% agarose gel.<br>
+<br>
+We made samples as follows.
 <Table Border Cellspacing="0">
 <Tr><Td>　　</Td><Td>　pDONR　</Td><Td>　BP TNFA-1　</Td><Td>　BP TNFA-2　</Td></Tr>
@@ Line 172: / Line 741: @@
 </Table>
 <br>
+<br>
+We electrophoresed them in following order.<br>
-<br><br>
+Left marker 5uL control pDONR DNA ,pENTR-TNFAIP3-1 DNA,pENTR-TNFAIP3 -2 DNA marker 4uL marker 3uL Wright.<br>
+<br>
+<strong>Results</strong><br>
 <img src="https://static.igem.org/mediawiki/2012/2/2e/0807.png" width="500" height="300">
-<br><br>
+<br>
+Concentration of pENTR-TNFAIP3-2 DNA was estimated at around 100ng/uL.<br>
+<br>
+<br>
+②　LR reaction<br>
+We made following solution(vials)in 1.5mL tube.<br>
 <Table Border Cellspacing="0">
@@ Line 187: / Line 760: @@
 <Tr><Td>　total　</Td><Td>　9μL　</Td></Tr>
 </Table>
+<br>
+we added 1uL of LR clonaseⅡ enzyme mix to this solution and incubate it for 2.5 hours.<br>
+<br>
+③ Transformation of E.coli with LR reaction products.<br>
+Transformation of E.coli was carried out by adding 2uL of LR reaction products to 100uL of XL1-Blue and plated on the LB ampicillin(+)plate and cultured at 37°C.<br>
+<br>
 <br>
 WEEK１終わり
 <br>
@@ Line 200: / Line 785: @@
 <h2>August 8th</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
+We isolated 5 colonies from the LB plate, and cultured.<br>
+<br>
+<br>
 <h2>August 9th</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
+①Purification of the candidate pUAS-flag-TNFAIP3 DNA<br>
+<br>
+The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.<br>
+We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.<br>
+<br>
+<br>
+②Characterization of the candidate pUAS-flag-TNFAIP3 DNA<br>
+<br>
+The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.<br>
+<br>
 <Table Border Cellspacing="0">
-<Tr><Td>　DNA sample　</Td><Td>　1μL　</Td></Tr>
+<Tr><Td>　Each diluted DNA sample　</Td><Td>　1μL　</Td></Tr>
 <Tr><Td>　10×H buffer　</Td><Td>　0.5μL　</Td></Tr>
 <Tr><Td>　EcoRⅠ　</Td><Td>　0.2μL　</Td></Tr>
@@ Line 219: / Line 815: @@
 <br>
 <br>
+After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.<br>
+Left,　10fold dilution,　2uL marker,　20fold dilution,　30fold dilution,　1uL marker,　40fold dilution,　0.5uL marker right.<br>
+<br>
+<br>
+<strong>Results</strong><br>
 <img src="https://static.igem.org/mediawiki/2012/9/9b/0809.png" width="500" height="300">
 <br><br>
+The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.<br>
+<br>
+<br>
+③ PCR amplification of the insert DNA<br>
+<br>
+The four 5'primers were designed for PCR as follows.<br>
+<br>
+・GW3r<br>
+’-ATCGAGGCCTGTCTAGAGAAGC<br>
+・TNFA-1<br>
+’-GGCTTTGCTATGATACTCGGAACTG<br>
+・TNFA-2<br>
+’-GTAAAATGTGAAACGCCCAACTGC<br>
+・TNFA-3<br>
+’-GGACTCCAGAAAACAAGGGCTTT<br>
+<br>
+<br>
+The 3’ primer was designed as follows.<br>
+・SVr<br>
+’-GGCATTCCACCACTGCTCCC<br>
+<br>
+<br>
+PCR reactions with the following combinations of primers were carried out.<br>
+sample１→GW3r―SVr<br>
+sample２→TNFA-1―SVr<br>
+sample３→TNFA-2―SVr<br>
+sample４→TNFA-3―SVr<br>
+<br>
+<br>
+PCR was carried out in the following reactions.
 <Table Border Cellspacing="0">
-<Tr><Td>　　</Td><Td>　1-2　</Td></Tr>
+<Tr><Td>　　</Td><Td>　Each sample　</Td></Tr>
 <Tr><Td>　50ng/μL LR TNFA-1　</Td><Td>　1μL　</Td></Tr>
 <Tr><Td>　10×rTaq buffer　</Td><Td>　2μL　</Td></Tr>
@@ Line 234: / Line 875: @@
 <Tr><Td>　total　</Td><Td>　20μL　</Td></Tr>
 </Table>
-<br>
+<br><br>
+Reaction conditions of PCR
 <Table Border Cellspacing="0">
 <Tr><Td>　temperature　</Td><Td>　time　</Td><Td>　cycle　</Td></Tr>
@@ Line 253: / Line 894: @@
 <h2>August 10th</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
-<br><br>
+Electrophoresis of PCR products was carried out.<br>
+After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.<br>
+<br>
+Left　marker5uL　sample１　sample２　sample３　sample４　Wright<br>
+<br>
+<strong>Results</strong><br>
 <img src="https://static.igem.org/mediawiki/2012/8/84/0810.png" width="500" height="300">
-<br><br>
+<br>
+The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.<br>
+<br>
+<br>
@@ Line 263: / Line 919: @@
 <h2>August 13th</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
+Cut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction.<br>
+<br>
+The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.<br>
+<br>
 <Table Border Cellspacing="0">
-<Tr><Td>　1-1　</Td><Td>　1μL　</Td></Tr>
+<Tr><Td>　the candidate pENTR-TNFAIP3 prepared on 8/6　</Td><Td>　1μL　</Td></Tr>
 <Tr><Td>　10×M buffer　</Td><Td>　0.5μL　</Td></Tr>
 <Tr><Td>　Hind Ⅲ　</Td><Td>　0.2μL　</Td></Tr>
@@ Line 275: / Line 935: @@
 </Table>
 <br>
+<br>After reaction, samples were electrophoresed in following order.<br>
+<br>
+Left: 1kb marker5uL cut sample <br>
+Right: uncut sample<br>
+<br>
+<br>
+<strong>Results</strong>
 <br>
 <img src="https://static.igem.org/mediawiki/2012/2/2a/0813.png" width="500" height="300">
 <br><br>
+Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful.<br>
+<br>
+<br>
 <h2>August 14th</h2>
 <br>
-<strong>*TNFA and API2</strong>
+<br>
-<br><br>
+Based on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again.<br>
+<br>
+Composition of the reaction
 <Table Border Cellspacing="0">
-<Tr><Td>　1-1　</Td><Td>　sample1　</Td></Tr>
+<Tr><Td>　　</Td><Td>　sample1　</Td></Tr>
 <Tr><Td>　10ng/μL TNFAIP3　</Td><Td>　6μL　</Td></Tr>
 <Tr><Td>　10×KOD plus buffer　</Td><Td>　10μL　</Td></Tr>
@@ Line 299: / Line 971: @@
 <Tr><Td>　total　</Td><Td>　100μL　</Td></Tr>
 </Table>
+<br>
 <br>
+Reaction conditions
 <Table Border Cellspacing="0">
 <Tr><Td>　temperature　</Td><Td>　time　</Td><Td>　cycle　</Td></Tr>
@@ Line 313: / Line 986: @@
 <br>
 <br>
+PCR product was applied to the 0.7% agarose gel electrophoresis.<br>
+<br>
+<strong>Results</strong><br>
 <img src="https://static.igem.org/mediawiki/2012/c/c4/0814.png" width="500" height="300">
-<br><br>
+<br>
+<br>
+<br>
 WEEK2終わり
@@ Line 335: / Line 1,014: @@
 <br>
 <h2>August 20th</h2>
-<br>
-<strong>TNFA and API2</strong>
 <br><br>
@@ Line 404: / Line 1,081: @@
 </div>
 </html>
+<UL>
+	<LI><P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><B>
+	</B></FONT><FONT FACE=""><SPAN LANG="en-US"><B>DNA purification and
+	precipitation</B></SPAN></FONT></P>
+</UL>