Difference between revisions of "Team:Tuebingen/Experiments"
m |
|||
Line 9: | Line 9: | ||
<div id="dia0" class="dia"> | <div id="dia0" class="dia"> | ||
− | <label class="collapse" for="_1"><h4> | + | <label class="collapse" for="_1"><h4>3A-Assembly</h4></label> |
<input id="_1" type="checkbox"> | <input id="_1" type="checkbox"> | ||
<div><ul> | <div><ul> |
Revision as of 16:53, 17 September 2015
<
>
- 1μl 10x ligase buffer
- 0,5μl ligase
- 1μl ATP
- 0,51μl linearised vector
- 33,5μl insert 1
- 33,5μl insert 2
Method:
- Mix all components
- Incubate overnight at 4°C
- Gelpurify using agarose gels
- autoclave 250mL LB in Erlenmeyer beaker
- plate cells from cell stock on agarose plate with appropriate antibiotics
- inoculate one clone in 5mL of LB with antibiotics, grow at 37°C o/n
- expand to 500mL and grow until OD600nm = 0.35
- transfer into 50mL Falcon tubes
- refrigerate centrifuge at 4°C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol
- spin down cells at 2000g for 10Min at 4°C
- resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2
- spin down at 2000g for 10Min at 4°C
- resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol
- freeze 50-100μl aliquots
- store at 20°C
- 0,1μl forward primer (10μM)
- 0,1μl reverse primer (10μM)
- 5μl 2x Q5 Mastermix
- 4,8μl H2O
- Pick a colony from plate and streak in a PCR tube. Mix components and add to tube.
- Run PCR:
- 95°C 5:00min
- 95°C 0:45min
- 53°C 0:45min
- 72°C 1:30min
- 72°C 10:00min
- 4°C inf
- 2μl 10x buffer (corresponding to enzymes)
- 0,5μl restriction enzyme 1
- 0,5μl restriction enzyme 2
- 1μg DNA
- fill up to 20μl with H2O
Method:
- mix all components
- incubate at 37°C for 2h
- heatinactivate enzymes at 80°C for 10min or use for gel purification
- Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube
- Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060°C until gel slice is completely dissolved
- Insert SV Minicolumn into Collection Tube
- Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute
- Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube
- Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min
- Discard flowthrough and reinsert Minicolumn into Collection tube
- Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min
- Empty the Collection Tube and centrifuge the column assembly for 1,5 min
- Leave the tubes open for 10 min (to let any rest of ethanol evaporate)
- Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube
- Add 30μl of NucleaseFreeWater (65°C) to the Minicolumn. Incubate at room temperature for 10 min
- Centrifuge at 16,000xg for 1 min.
- Incubate sample at 65°C for 5min
- Discard Minicolumn and store DNA at 4°C or 20°C
This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega
- 1μl 10x ligase buffer
- 0,5μl ligase
- 1μl ATP
- 0,51μl linearised vector
- 7μl insert
Method:
- Mix all components
- Incubate overnight at 4°C
- Gelpurify using agarose gels
MutagenesisPCR
- 200ng template
- 1μl forward primer (2mM)
- 1μl reverse primer (2mM)
- 1μl Phu polymerase
- 1μl dNTPs
- 10μl 5x Buffer
- to 50μl with H2O
- Mix components
- Run thermocycler:
- 98°C 0:30min
- 98°C 0:45min
- 55°C 0:45min
- 72°C 7:00 min
- 72°C 1:00 min
- 4°C inf
Oligo Annealing
- 2μl oligo 1
- 2μl oligo 2
- 96μl elution buffer
Method:
- Mix all components
- incubate for 10 minutes at 95°C
- take tube with heat block out of heat and let it cool down to room temperature
Preparation was done using the PureYield™ Plasmid Miniprep System by Promega´
- Pick colonies for overnight cultures in 23ml LB medium with antibiotic grow overnight
- Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1,5ml, spin again and discard supernatant
- Resuspend the bacteria pellet in 600μl H2O
- Add 100 μl Cell Lysis Buffer, invert six times
- Add 350 μl cold Neutralization Solution, mix thoroughly by inverting
- Centrifuge 30s at maximum speed
- Transfer supernatant to PureYield Minicolumn in collection tube
- Centrifuge for 30 sec, discard the flow-through
- Add 200 μl Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.
- Add 400 μl Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.
- Place column in eppendorf tube, add 30 μl Elution Buffer to minicolumn matrix, incubate for 1 min
- Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA
- Measure the DNA concentration using a Nanodrop
- 0,25μl Enzyme 1
- 0,25μl Enzyme 2
- 1μl 10x buffer (corresponding to enzymes)
- 200-300ng DNA
- Fill up with water to 10μl.
Method:
- Incubate at 37°C for 1h
- Run on 1% agarose gel
- 50μl chemically competent E. coli
- 35μl overnight ligation mix OR 1ng purified plasmid DNA
Method:
- Thaw bacteria pellet on ice
- add ligation mixture or purified plasmid to bacteria
- incubate for 20min on ice
- heat shock bacteria for 45s at 42°C
- incubate bacteria on ice for 2min
- add 700μl LB medium without antibiotics
- incubate for 1h at 37°C
- spin down bacteria at 2g for 2min
- discard supernatant by tipping the tube
- resuspend bacterial pellet in leftover supernatant (50100μl)
- streak bacteria onto LBagar plates with antibiotics and incubate overnight
- ca. 10ml YPD
- 100μl OneStep buffer
- 20μg ssDNA
- 100-500 ng plasmid DNA
- fresh YPD selective plate
Method:
- Scrape some yeast cells off a fresh YPD plate and inoculate in liquid YPD (= culture).
- Thaw ssDNA (e.g. salmon sperm DNA) and heat for 10 min at 95 °C, then chill on ice.
- Pellet 1 mL of culture by centrifugation at > 13.000 rpm.
- Discard supernatant and resuspend cells in 100 μL OneStep buffer, vortex heavily
- Add 20 μg ssDNA (10 μL of 2 mg/mL) and 100 500 ng plasmid DNA to be transformed
- Vortex and incubate at 45 °C for 2 h
- Add 1 mL YPD, mix and spin 10 sec at full speed, discard supernatant
- Resuspend cell pellet in 1000 μL YPD and plate 100 μL directly on appropriate selective plates
- Colonies appear after 2 days of incubation at 30 °C
- 1M tris
- 0,85M B(OH)3
- 2mM EDTA
- pH=8,0
- 20mM Tris/HCl pH 7,6
- 250mM Glycin
- 0,1% (w/v) SDS
- in ddH2O
- 50mM Tris pH 6,8
- 1,25mM EDTA pH 8
- 12,5% (v/v) Glycin
- 2% (w/v) SDS
- 50mM DTT
- 2,5% (v/v) -Mercaptoethanol
- 0,025% (w/v) Bromphenolblau
- in ddH2O
- 20g Lennox Broth
- to 1l with H2O
- 35g LB-Agar (Lennox)
- to 1l H2O
- 2g yeast nitrogen base w/o amino acids
- 0,25g synthetic complete drop-out mix
- 16,5mg adenine-sulfate
- to 300ml H2O
- adjust pH to 5.6
- add agar if needed
- 1g adenine hemisulfate
- 1g arginine-HCl
- 1g histidine HCl*
- 1g isoleucine
- 2g leucine*
- 2g lysine-HCl
- 2g methionine*
- 1,5g phenylalanine
- 1g serine
- 1g threonine
- 1,5g tryptophane*
- 1g tyrosine
- 0,6g uracil*
- 4,5g valine
- for dropout mix, omit appropriate components, labelled with *
- combine ingredients and mix thoroughly
- 3g yeast extract
- 6g peptone
- 30mg adenine hemisulphate
- 300ml H2O
- if needed, add 5g agar
- 20g of appropriate sugar
- 100ml H2O
- CA 34µg/ml
- Amp 100µg/ml
- 0,2M LiAc
- 40% PEG4000
- 100mM DTT
- sterile filtrated
SDS-Gels
- 40,5% acrylamide (30%)
- 0,375M Tris (pH 8,8)
- 1% SDS (10%)
- 1% APS(10%)
- 0,1% TEMED
- 17% acrylamide (30%)
- 0,125M Tris (pH 6,8)
- 1% SDS (10%)
- 1% APS (10%)
- 0,1% TEMED
Ni-NTA buffers
All buffers are from the Qiagen "Ni-NTA Agarose Purification of 6xHis-tagged Proteins from E. coli under Native Conditions - Quick-Start"-protocol.
- 50 mM NaH2PO4
- 300 mM NaCl
- 10 mM Imidazole
- 50 mM NaH2PO4
- 300 mM NaCl
- 20 mM Imidazole
- 50 mM NaH2PO4
- 300 mM NaCl
- 250 mM Imidazole
Experiments
Describe the experiments, research and protocols you used in your iGEM project.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project