Difference between revisions of "Team:Kyoto/MaterialsMethods"

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<h6 id = "Plates">4-10 Plates</h6>
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<p>
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1.Rinse the graduated cylinder with distilled water and add 500 ml of milliQ.
 +
<br>2. Transfer 1. to the flask.
 +
<br>3. Place the following in the flask of 2.
 +
<br>  ・Bacto Tryptone 5 g
 +
<br>  ・Bacto yeast extract 2.5 g
 +
<br>  ・NaCl 2.5g
 +
<br>  ・Agar 7.5 g
 +
<br>4. After mixing thoroughly, autoclave it after covering with aluminum foil.
 +
<br>5. Add the respective antibiotics.
 +
<br>6. Cool until it gets moderately warm to the touch.
 +
<br>7. Put in a container and fix the lid with the tape after the medium solidifies.
 +
</p>
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<table>
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<h7> Concentrations of antibiotics:</h7>
 +
<tr><th>Antibiotics</th><th>Stock concentration</th><th>Final concentration</th></tr>
 +
<tr><td>Ampicillin</td><td>100mg/ml</td><td>100μg/ml</td></tr>
 +
<tr><td>Chloramphenicol</td><td>10mg/ml</td><td>30μg/ml</td></tr>
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</table>
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Revision as of 13:31, 26 September 2018

Material&Methods

1) Parts
Basic Parts
Composite Parts
2) Primer List
primer namesequenceLengthGC%TmDesignerManufacturer
Z001GTTAGGGCAGGGATGTAGATT2147.6253.07Shimazoeeurofin
Z002TGGTTAACGTATTCTCGATGTAAAG253653.19Shimazoeeurofin
Z003TGGTTACAAACTACCTACAATTTG2433.3351.29 Shimazoeeurofin
Z004GATCTTTTACCTGATTTCGACC2240.9151.18Shimazoeeurofin
Z005TGTTCACACTTAATTCACATTTATTTGAGGCAACAATACGTGGCCAGCGACATGGAGGCCCAGAA6544.6272.86Shimazoeeurofin
Z006ATGAATAAGGAAAAAGATAGGGAGCACTTAATAGGCCCTGCCCTCGTTTAAACTGGATGGCGG6346.0371.92Shimazoeeurofin
Z007acacgctttttcagttcgagtttat253655.89Shimazoeeurofin
Z008gtttcgaataaacacacataaacaaacaaaatgcgtaaaggagaagaacttttcactgga6033.3366.8Shimazoeeurofin
Z009tccagtgaaaagttcttctcctttacgcattttgtttgtttatgtgtgtttattcgaaac6033.3366.8Shimazoeeurofin
Z010catggcatggatgaactatacaaataataaAACAGGTGGATCCCACATTGtcatgtaatt603566.71Shimazoeeurofin
Z011aattacatgaCAATGTGGGATCCACCTGTTttattatttgtatagttcatccatgccatg603566.71Shimazoeeurofin
Z012ggccgcaaattaaagccttcgagcg255663.35Shimazoeeurofin
Z013gtttcgaataaacacacataaacaaacaaaatggcttcctccgaagacgttatcaaagag6036.6767.57Shimazoeeurofin
Z014ctctttgataacgtcttcggaggaagccattttgtttgtttatgtgtgtttattcgaaac6036.6767.57Shimazoeeurofin
Z015aataacgctgatagtgctagtgtagatcgcAACAGGTGGATCCCACATTGtcatgtaatt6041.6769.94Shimazoeeurofin
Z016aattacatgaCAATGTGGGATCCACCTGTTgcgatctacactagcactatcagcgttatt6041.6769.94Shimazoeeurofin
Z017gaattcgcggccgcttctagagaca255662.89Shimazoeeurofin
Z018tgccggactgcagcggccgctacta256869.57Shimazoeeurofin
Z019AAACATACTATTTAGGCTTGTTTATGTTCAGAACCTGTGACTCGTTTAAACTGGATGGCGGCGTT654070.47Shimazoeeurofin
Z020atggccgctactgacagattaaacc254858.98Shimazoeeurofin
Z021gagacccatcttgtaactcaatacg254455.16Shimazoeeurofin
Z022gaataaacacacataaacaaacaaaatggccgctactgacagattaaacc503665.41 Shimazoeeurofin
Z023ggtttaatctgtcagtagcggccattttgtttgtttatgtgtgtttattc503665.41Shimazoeeurofin
Z024cgtattgagttacaagatgggtctcCACCACCACCACCATCACtagAACAGGTGGATCCCACATTGtcatg7149.373.64Shimazoeeurofin
Z025catgaCAATGTGGGATCCACCTGTTctaGTGATGGTGGTGGTGGTGgagacccatcttgtaactcaatacg7149.373.64Shimazoeeurofin
Z026gaattcgcggccgcttctagagacacgctttttcagttcgagtttat4746.8169.83Shimazoeeurofin
Z027tgccggactgcagcggccgctactagtaggccgcaaattaaagccttcgagcg5360.3877.31Shimazoeeurofin
Z028TGAAAACTCATTACCTAAATTTGTTTATGTTCGGTAGCCCCTCGTTTAAACTGGATGGCGGCGTT6541.5471.34Shimazoeeurofin
Z029GTATCCTTTTCAAGTACTTCCACCACCACCACCATCACta404565.52Shimazoeeurofin
Z030taGTGATGGTGGTGGTGGTGGAAGTACTTGAAAAGGATAC404565.52Shimazoeeurofin
Z031caacctcaatggagtgatgcaacc245058.35Shimazoeeurofin
Z032AACAGGTGGATCCCACATTGtc225056.65Shimazoeeurofin
Z033CCTTTGCTCTGACCGATCCATA225056.03Shimazoeeurofin
Z034ACCCAGCACCATCAGAATTTAGCG245059.41Shimazoe eurofin
Z035CGGTGATTATCTAATCGAGGAAGAGG2646.1556.42Shimazoeeurofin
Z036GCTCATCAGTTCAATGGGAATCTTG254456.33Shimazoeeurofin
Z037CAGCTTTGCTGCTATGTATGGTG2347.8356.35Shimazoeeurofin
Z038GGAACCGCAAAACCAGACTAC2152.3855.81Shimazoeeurofin
Z039tttgtttgtttatgtgtgtttattcgaaac3026.6754.96Shimazoe eurofin
Z040AACAGGTGGATCCCACATTGtcatgtaatt304060.27Shimazoeeurofin
Z041gtttcgaataaacacacataaacaaacaaa3026.6754.96Shimazoeeurofin
Z042aattacatgaCAATGTGGGATCCACCTGTT304060.27Shimazoeeurofin
Z043GAAATTGCACTACCACCGGCGGCAAAATAT3046.6763.65Shimazoeeurofin
3) Materials
3-1 Kit
NameSupplier
FastGene™Plasmid Mini KitNIPPON Genetics Co., Ltd
FastGene™Gel/PCR Extraction Kit NIPPON Genetics Co.,Ltd
3-2 Restriction Enzyme
NameSupplier
EcoRITaKaRa, Promega
PstITaKaRa, Promega
SpeITaKaRa, Promega
3-3 Polymerase
NameSupplier
Q5 High-Fidelity 2x master mixNew England Biolabs Japan Inc.
3-4 DNA ligase
NameSupplier
T4 DNA ligaseTaKaRa
3-5 Marker
NameSupplier
1kb DNA LadderNIPPON Genetics Co., Ltd
3-6 Organism
NameSupplier
E.coli DH5α GenotypeTaKaRa
3-7 Antibiotics
NameSupplier
ChloramphenicolWako
AmpicillinWako
3-8 Equipment
Name Supplier
BioPhotometereppendorf
LABO SHAKERBIO CRAFT
CO2 INCUBATORSANYO
Pipette ControllerBiohit Midi Plus
MiniCentrifuge Model GMC-060LMS CO.,LTD.
HIGH-SPEED REFRIGERATED CENTRIFUGETOMY
HIGH-PRESSURE STEAM STERILIZERTOMY
VOTEX-GENE2Scientific Industryes
Scanning Electron Miniscope TM1000sHITACHI
Fluorescence Microscope BX61N-34-FL-1-DOLYMPUS
SCIECE IMAGING SYSTEM LAS-3000Fuji film
3-9 Backbone
NameSupplier
pRS421National BioResource Project
http://yeast.nig.ac.jp/yeast/
pRS423 National BioResource Project
pRS424National BioResource Project
pRS425 National BioResource Project
pRS426National BioResource Project
pGK421National BioResource Project
pRS316 National BioResource Project
3-10 Buffer
NameSupplier
Sodium CarbonateWako
Sodium BicarbonateWako
Methanol(99.5%)Wako
Sodium ChlorideWako
SDSnacalai tesque
Trisaminomethanenacalai tesque
Potassium dihydrogenphosphsteSIGAMA-ALDRICH
Potassium ChlorideWako
GlycineWako
Agar, PowderWako
Agarose XPWako
10% Hydrochloric AcidWako
3-11 SDS-PAGE&Westernblotting
NameSupplier
REAL GEL PLATE (10%)BIO CRAFT
BE-210(SDS-PAGE)BIO CRAFT
BE-330BIO CRAFT
Amersham ECL Anti-Mouse IgGGE healthcare
Amersham ECL Anti-rabbit IgGGE healthcare
Amersham ECL Prime Blocking ReagentGE healthcare
Amersham ECL Prime WB Detection ReagentGE healthcare
4) Methods
4-1 Miniprep

Minipreps were performed using FastGene™Plasmid Mini Kit according to the manufacturer's protocols.

4-2 Gel Extraction and PCR purification

Gel Extraction was performed using FastGene™Gel/PCR Extraction Kit according to the manufacturer's protocols.

4-3 Restriction Enzyme Digestion

Restriction enzyme treatment was performed using Q5 High-Fidelity 2x master mix according to the respective manufacturer's protocols.

4-4 Ligation

Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's.

4-5 Transformation

1.Thaw competent cells on ice
2.Add DNA sample (2.5μl) to competent cells. leave them on ice for 15 minutes
3.Keep them in heating block for 45 seconds, then cool on ice for 2 minutes
4.Add 90μl SOC liquid culture medium, then incubate for 45 minutes at 37℃
5.Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37℃

4-6 PCR

PCR was performed using Q5 High-Fidelity 2x master mix according to the manufacturer's protocols.

4-7 Sequencing

We outsourced the sequencing to Macrogen

http://www.macrogen-japan.co.jp/cap_seq_0203.php

4-8 Western blotting

1.Apply sample to SDS-PAGE minigel (BIOCRAFT 15%).
2.Soak the gel in transfer buffer.
3.Soak PVDF membrane in 100% methanol for 30 sec.
4.Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min.
5.Set filter paper, membrane, gels, filter paper in this order to semidry transfer from anode to cathode.
6.Transfer the proteins from the gel to the membrane with 100 mA for 1 h.
7.Soak membrane in blocking buffer (dilution of 5 g ECL Blocking reagent by 100ml of TBST) for 1 h.
8.Wash for 5 min 3 times with TBST.
9.Apply 1' antibody (dilution of 10 μl of 1’antibody by 10ml of Blocking buffer) and shake for 1 h.
10.Wash for 5 min 3 times with TBST.
11.Apply 2' antibody (dilution of 4μl of 2’antibody by 10ml of Blocking buffer) and shake for 1 h.
12.Wash for 5 min 3 times with TBST.
13.Drain excess wash buffer from the washed membrane and place on flat surface, protein side up.
14.Add detection reagent onto the membrane, covering all of the membrane.
15.Incubate for 5 minutes at room temperature.
16.Drain off excess detection reagent by dabbing with Kimwipe.
17.Place the sample in the CCD camera compartment and record the images.

4-9 Overnight-Culture

1.Prepare LB media by resolving 5g Bacto Tryptone, 1.25g Bacto-yeast extract and 1.25g NaCl in 500ml of milliQ and autoclave it.
2.Transfer 7ml of LB into a culture tube and add the respective antibiotic stock solutions.
3.Inoculate the culture by picking a single colony with a pipet tip and tipping it into the medium.
4.Shake the culture at 37℃ at 180 rpm for 14-16h.
5.Isolate plasmids or use the culture for further experiments.

Concentrations of antibiotics:
AntibioticsStock concentrationFinal concentration
Ampicillin100mg/ml100μg/ml
Chloramphenicol10mg/l30μg/ml

4-10 Plates

1.Rinse the graduated cylinder with distilled water and add 500 ml of milliQ.
2. Transfer 1. to the flask.
3. Place the following in the flask of 2.
・Bacto Tryptone 5 g
・Bacto yeast extract 2.5 g
・NaCl 2.5g
・Agar 7.5 g
4. After mixing thoroughly, autoclave it after covering with aluminum foil.
5. Add the respective antibiotics.
6. Cool until it gets moderately warm to the touch.
7. Put in a container and fix the lid with the tape after the medium solidifies.

Concentrations of antibiotics:
AntibioticsStock concentrationFinal concentration
Ampicillin100mg/ml100μg/ml
Chloramphenicol10mg/ml30μg/ml