Difference between revisions of "Team:Tuebingen/Experiments"

Protocols

MiniPrep

• Resuspension of the bacteria pellet (from 3ml culture) in 600μl H2O.
• Add 100 μl cell Lysis Buffer, invert it.
• Add 350 μl cold Neutralization Solution, invert the mixture.
• Centrifuge 5 min. at max. speed.
• Transfer supernatant in PureYield Minicolumn in Collection Tube.
• Centrifuge for 30 sec, discard the flowthrough.
• Add 200 µl Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flowthrough.
• Add 400 µl Column Wash Solution(CWC), centrifuge for 30sec, discard the flowthrough.
• Place column in reaction vessel, add 30 μl Elution Buffer to minicolumn matrix, incubate 1 min at 37°C.
• Centrifuge the mixture.
• Measurement of the DNA concentration at the Nanodrop

Colony-PCR

• Pick a colony from plate and strike out in PCR tubes.
• Add 5μl Q5 High-Fidelity 2X MasterMix from NEB.
• Add 1,8μ of each primer (1μMol): fw/rv
• Add x μl water up to 10μl per mixture.

3A-Assembly

Control Restriction

• 0,2μl EcoRI-HF
• 0,2μl PstI
• 1μl Buffer
• 250ng DNA[?]
• Fill up with water to 10μl.
• Incubate at 37°C for 1,5h.
• Run 1% agarose gel

Transformation

• Take 50μl competent cells and 5μl ligation/plasmid.
• Incubate at 37°.C
• Centrifuge at 2000xg for 2 min.
• Discard the supernatant, resuspend the rest and do the plating.

PCR

Ligation

• 5 μl Water
• 2 μl Puffer NEB
• 1 μl Ligase NEB
• 2 μl Plasmid (pSB)
• 10 μl Insert
• 4 °C o/n

1% Agarose gel

• 3 μl Midori green
• 100 ml TBE
• 1g Agarose

Pouring agar plates

• LB Agar-Agar
• 200ml 1:1000 Chloramphenicol(11 plates)
• 200ml 1:1000 Ampicillin(11 plates)

Gel extraction / DNA Purification using Promega Wizard SV Gel and PCR Clean-Up System Kit

This is a modified protocol for the Wizard SV Gel and PCR Clean-Up 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 50-60°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 recentrifuge the column assembly for 1,5 min.
• Leave the tubes open for 10 min (to let any rest of ethanol evaporate).
• Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.
• Add 30μl of Nuclease-Free-Water (65°C) to the Minicolumn. Incubate at 65°C for 5 min. Centrifuge at 16,000xg for 1 min.
• Discard Minicolumn and store DNA at 4°C or -20°C.

Buffers and Media

TBE

• 1M tris
• 0,85M B(OH)3
• mM DTA
• 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