Team:Tuebingen/Methods


  • 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
Method:
  • 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

    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
  • 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
  • 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
  • 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
  • chloramphenicol 34µg/ml
  • ampicillin 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

  • cast gels
  • load samples with loading dye
  • run gels at 120 V
  • pETue-NAGA-Intein was transformed into E. coli BL21(DE3)
  • grow culture to OD_600 = 0.3
  • induce with 0.2 mM IPTG
  • express O/N at room temperature
  • centrifuge at 6000 g for 30 minutes at 4°C
  • resuspend pellet in 10 ml lysis buffer
  • sonicate for a total of 3 minutes (40% amplitude, 1 s intervals)
  • Cells were measured undiluted or in 1:2 dilution in 96 well plates (black, flat bottom)
  • Following excitation and emission wavelengths were used for the different fluorescent proteins:
  • GFP
    • Exc: 488 nm
    • Em: 520 nm
  • Dronpa
    • Exc: 503 nm
    • Em: 535 nm
  • RFP
    • Exc: 584 nm
    • Em: 616 nm
  • Fluorescence was measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10

    Sample preparation

  • A thin layer of low melting agarose was supplied on glass slides
  • 10 µl Yeast cells grown overnight were inoculated in the solidified agarose
  • samples were used for microscopy after at least 30min incubation at 30° in a humid environment

    • Microscopy Setup

  • We used a Zeiss LSM 710 microscope with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. All pictures were taken with one active fluorescent channel and brightfield channel. Dronpa was observed by using a 514 nm laser (2% power) for excitation, and RFP was observed by using a 633 nm laser (2% power) for excitation

    • Dronpa Illumination

  • Illumination of dronpa was performed in vivo using a 488 nm argon laser at 50% power for off switching and a 405 nm for on switching of fluorescence. The illumination of cells was either performed using continuous illumination for 10 to 20 seconds or by performing a line scan with the illumination laser.
  • Grow overnight culture of yeast: Induction or repression of luciferase expression by addition of different galactose/glucose/raffinose concentrations for pGal or pSUC promoters.
  • NanoLuc luciferase protocol: add 25 µl cell culture, 25 µl reconstituted luciferase assay reagent and 50 µl PBS in 96 well plate (white, clear bottom)
  • Firefly luciferase protocol: mix either cell suspension or cell lysate 1:1 with reconstituted luciferase assay reagent
  • Luciferase activity measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10.
  • incubate 100 µl of Ni-NTA purified eluate with 10 µl (10 mg/ml, 7,90 mM) peptide solution for 24 or 48 hours at 5°C or room temperature
  • dilute with 110 µl of 2x Laemmli buffer
  • incubate at 95°C for 10 minutes
  • load onto SDS polyacrylamide gel
    Preparation and trypsin digest:
  • cut bands from SDS polyacrylamide gel
  • cover gel piece with Buffer2 for 30 min to discolor
  • cover with acetonitrile (ACN) for 10 min to dehydrate
  • cover with Buffer1 containing 10 mM DTT to reduce for 45 min
  • cover with Buffer1 containing 55 mM iodoacetamide (IAA) to alkylate for 30 min
  • cover with Buffer1 for 15 min to wash
  • cover with Buffer2 for 10 min to wash
  • cover with 100% ACN for 10 min to dehydrate
  • cover with 20 µl Buffer1 containing 100 ng Trypsin
  • digest for 5 h at 37 °C
  • stop reaction by addition of 2 µl of 10% TFA
  • extract peptides by incubation for 30 minutes
    • Buffer1: 50 mM ammoniumbicarbonat (AB) Buffer2: 70% 50 mM AB, 30% ACN Measurement:
  • mix 1 ml of sample with 1 µl of gentisic acid matrix solution
  • pipette unto target
  • wait for crystallisation
  • measure with MALDI-TOF
  • analyse data using FlexAnalysis und FlexControl
    • The result score is calculated as Score = -log10(P).
    Pellet
  • centrifugation of the bacterial culture for 10 min at 8000 rpm at 4 °C
  • discard the supernatant
    • Lysis
  • per 1g wet weight of the pellet add 4ml lysis buffer
  • fill up to 0.5 mg/ml with lysozyme
  • incubation on ice for 2 h
  • centrifuge for 30 min at 10000 g at 4 °C
    • Column
  • equilibrate the column with 0.5 ml Ni-NTA (0.25 ml bed volume)
  • add 0.5 ml lysis buffer and slightly centrifuge the buffer through the column, it has to stay wet (do twice)
  • pump the cell lysate (supernatant) for 1 h at ice through the column
    • Wash
  • twice with 1.25ml wash buffer
  • four times with 0.5 ml elution buffer
  • recover the supernatant
  • Load a sample of every step in an SDS gel.
  • centrifuge lysate at 15,000 g for 30 min
  • purify supernatant with Ni-NTA beads (native conditions, gravity flow)
  • elute once with 500 µl elution buffer
  • Cast gels with composition as described (Link bitte einfügen zu der Zusammensetzung)
  • Load samples with SDS-PAGE loading dye
  • Run gels at 150V until front is run through the gel