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Team:KIT-Kyoto/c6h12o6
From 2012.igem.org
Template:KIT-Kyoto-1 Template:KIT-Kyoto-header
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˚.
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Transfer the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute, and discard the supernatant (2 times).
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Add 250 µL of SolutionⅠ to the pellet and mix well with vortex.
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Add 250 µL of SolutionⅡ and mix well by turning the tube upside down several times.
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Add 350 µL of SolutionⅢ and by turning the tube upside down several times.
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Centrifuge at 13,000 rpm for 5 minutes.
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Transfer the supernatant (approx. 850µL) to a mini prep column.
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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.
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Add 500 µL of SolutionⅣ.
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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.
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Add 700 µL of SolutionⅤ.
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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through (2 times).
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Set the column on a new 1.5ml tube and add 100 µL of nuclease-free water.
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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.
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Suspend a small quantity of the colony in a tube using a sterilized toothpick.
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Incubate the tube at 65°C for 10 minutes.
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Add a drop of 10x Loading Buffer.
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Add equal volume of phenol/chloroform.
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Mix with vortex.
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Centrifuge at 13,000rpm for 3 minutes.
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Apply the supernatant to 1% agarose gel electrophoresis.
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Stain the gel in ethidium bromide solution for 10 minutes.
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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
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Apply the acrylamide solution mix to the PAGE glass plate.
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Deposit H2O carefully on the top of the acrylamide solution mix.
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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
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After the polymerization of the separation gel, remove the H2O of the top layer.
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Apply stacking gel mix on the running gel and put the comb to make wells.
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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.
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Add 450ul of H2O and 50 µL of FastBreak Cell Lysis Reagent (Madison, Wisconsin, USA Promega).
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Mix them for 15minutes with shaker.
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Add 100ul of 6ｘSDS-Sample buffer and mix with vortex.
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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%)
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Apply the samples and markers.
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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.
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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).
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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
