Team:Tuebingen/Experiments

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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.

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