Difference between revisions of "Team:Tuebingen/Experiments"

Protocols

3A-Assembly

• 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

Chemically Competent E.coli Cells

• 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

ColonyPCR

• 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

DoubleDigest Restriction of gBlocks and BioBricks

• 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

Gelpurification

This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega

• 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

Ligation

• 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

Smallscale plasmid preparation

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

Testrestriction

• 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

Transformation of Bacteria

• 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

Transformation of Yeast

• 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

Buffers and Media

TBE

• 1M tris
• 0,85M B(OH)3
• 2mM EDTA
• pH=8,0

SDS-PAGE running buffer

• 20mM Tris/HCl pH 7,6
• 250mM Glycin
• 0,1% (w/v) SDS
• in ddH2O

1x Laemmli

• 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

LB

• 20g Lennox Broth
• to 1l with H2O

LB/Agar

• 35g LB-Agar (Lennox)
• to 1l H2O

SC-media

• 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

synthetic complete drop-out mix

• 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

YEP (yeast extract peptone)

• 3g yeast extract
• 6g peptone
• 30mg adenine hemisulphate
• 300ml H2O
• if needed, add 5g agar

20% Glucose/Galactose/raffinose

• 20g of appropriate sugar
• 100ml H2O

antibiotics concentration

• CA 34µg/ml
• Amp 100µg/ml

One-step buffer

• 0,2M LiAc
• 40% PEG4000
• 100mM DTT
• sterile filtrated

SDS-Gels

12% separation gel

• 40,5% acrylamide (30%)
• 0,375M Tris (pH 8,8)
• 1% SDS (10%)
• 1% APS(10%)
• 0,1% TEMED

5% stacking gel

• 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.

Lysis buffer (pH = 8)

• 50 mM NaH2PO4
• 300 mM NaCl
• 10 mM Imidazole

Washing buffer (pH = 8)

• 50 mM NaH2PO4
• 300 mM NaCl
• 20 mM Imidazole

Elution buffer (pH = 8)

• 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

Inspiration

• 2014 Colombia
• 2014 Imperial
• 2014 Caltech