Team:Bielefeld-CeBiTec/Notebook/Protocols

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Protocols

Genetic engineering
PCR

PCR

  • Phusion (our standard) master mix (25 µl):
    • 5 µl 5 x HF-buffer
    • 0.5 µl 10mM dNTPs
    • Up to 20 ng * µl-1 template
    • 0.25 µl polymerase
    • 0.25-0.5 µl primer 10 µM (each one)
    • 0.75 µl DMSO (optional)
    • Fill up to 25 µl with H2O

  • Q5 master mix (25 µl):
    • 5 µl 5 x buffer
    • 0.5 µl 10 mM dNTPs
    • Up to 50 ng * µl-1 template
    • 0.1 µl polymerase
    • 0.5-1 µl primer 10 µM (each one)
    • Fill up to 25 µl with H2O

  • KOD master mix (50 µl):
    • 5 µl 10 x buffer for KOD Hot Start DNA Polymerase
    • 3 µl 25 mM MgSO4
    • 1 µl 10mM dNTPs
    • 1 µl polymerase
    • 1.5 µl primer 10 µM) (each one)
    • 10 ng * µl-1 template
    • Fill up to 50 µl with H2O
Colony PCR

Colony PCR

  • First alternative:
    • Pick one colony with a sterile tip and elute it in 100 µl ddH2O or medium
    • Store the colony at 4 °C while colony PCR is running
  • Second alternative:
    • Pick one colony with a sterile tip and streak cells at a marked position on a new plate
    • Put tip in PCR tube already containing the reaction mixture

    For both alternatives continue as follows:

  • One reaction mix contains:
    • 5 µl 5x buffer
    • 1 µl MgCl2 (25 mM stock)
    • 0.5 µl 10mM dNTPs
    • 0.25 µl primer mix (prefix/suffix primers or sequencing primers)
    • 17.625 µl ddH2O
    • 0.125 µl GoTaq polymerase (Promega)
    • 0.5 µl template
  • PCR program:
    • Cell lysis and initial denaturation: 5 min, 95 °C
    • 30 cycles of:
      • 10 s, 95 °C
      • 30 s, annealing temperature
      • 1 min / 1 kb of expected product, 72 °C
    • Final elongation: 5 min, 72 °C
  • Gel electrophoresis: check the fragment size

  • First (as above) alternative:
    • Plate the correct colony
  • Second (as above) alternative:
    • Use cells from the right positions to start liquid cultures or streak them on a new plate
DNA isolation (genomic DNA)

DNA isolation (genomic DNA)
  • The buffer and spin column for this DNA isolation were taken from the QIAprep® Spin Miniprep Kit

  • Resuspend the cells of the plate in 800 µl Buffer P1.
  • Transfer the suspension in small tubes with beads.
  • Use the ribolyser with 6200 rpm 3 x 60 seconds.
  • Centrifuge at top speed for 3 minutes.
  • Transfer 500 µl of the supernatant in a new 2 ml reaction tube.
  • Add 500 µl of Buffer P2 and mix thoroghly by inverting the tube 6-8 times.
  • Add 700 µl of Buffer N3 and mix thoroghly by inverting the tube 6-8 times.
  • Centrifuge at top speed for 10 minutes.
  • Apply the supernatant to the spin column by decanting or pipetting.
  • Centrifuge at top speed for 30-60 seconds and discard the flow-through.
  • Wash the spin column by adding 750 µl Buffer PE.
  • Centrifuge for 30-60 seconds and discard flow-through.
  • Wash again by adding 750 µl Buffer PE.
  • Centrifuge for 30-60 seconds and discard flow-through.
  • Centrifuge 2 minutes to remove residual wash buffer.
  • Place the column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 20 µl H2O, incubate at room temperature for a few minutes and centrifuge at top speed for 1 minute. Repeat this step.
DNA Purification

DNA Purification by Centrifugation

  • DNA purification by centrifugation by Promega
    • First alternative:
      • Dissolving the Gel Slice
        • Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 ml microcentrifuge tube.
        • Add 10 µl Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50-65°C until gel slice is completely dissolved.
    • Second alternative:
      • Processing PCR Amplifications
        • Add an equal volume of Membrane Binding Solution to the PCR amplification.

    • Binding of DNA
      • Insert SV Minicolumn into Collection Tube.
      • Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
      • Centrifuge at 16,000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
    • Washing
      • Add 700 µl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
      • Repeat Step before with 500 µl Membrane Wash Solution. Centrifuge at 16,000 x g for 5 minutes.
      • Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
    • Elution
      • Carefully transfer Minicolumn to a clean 1.5 ml microcentrifuge tube.
      • Add 15 µl of Nuclease-Free Water to the Minicolumn. Incubate at 60°C for 5 minutes. Centrifuge at
        16,000 x g for 1 minute. Repeat this step.
      • Discard Minicolumn and store DNA at 4°C or -20°C.

  • QIAquick Gel Extraction Kit by QIAGEN
    • Note: All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge.

    • Excise the DNA fragment from the agarose gel with a clean, sharp scapel.
    • Wigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100 µl). The maximum amount of gel per spin column is 400 mg. For > 2% agarose gels, add 6 volumes Buffer QG.
    • Incubate at 50 °C for 10 min (or until the gel slice has completely dissolved). Vortex thee tube every 2-3 min to help dissolve the gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
    • Add 1 volume isopropanol to the sample and mix.
    • Place a QIAquick spin colun in a provided 2 ml collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 µl, load and spin again.
    • If the DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 µl Buffer QG to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAqick column back into the same tube.
    • To wash, add 750 µl Buffer PE o QIAquick column and centrifuge for 1 mi. Discard flow-through and place the QIAquick column back into the same tube.
      Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt-ended ligation), let the column stand 2-5 min after addition of Buffer PE.
      Centrifuge the QIAquick clumn in the provided 2 ml collection tube 1 min to remove residual wash buffer.
    • Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
    • To elute DNA, add 50 µl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAqick membrane, increasing the incubation time up to 4 min can increase the yield of purified DNA.
    • If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
Plasmid Isolation

Plasmid isolation

  • Wizard® Plus SV Minipreps DNA Purification System by Promega (Our standard kit)
    • Production of cleared lysate:
      • Pellet 1-10 ml of overnight culture for 5 minutes
      • Thoroughly resuspend pellet with 250 µl of Cell Resuspension Solution
      • Add 250 µl of Cell Lysis Solution to each sample; invert 4 times to mix
      • Add 10 µl of Alkaline Protease Solution; invert 4 times to mix. Incubate 5 minutes at room temperature
      • Add 350 µl of Neutralization Solution; invert 4 times to mix
      • Centrifuge at top speed for 10 minutes at room temperature
    • Binding of plasmid DNA
      • Insert Spin Column into Collection Tube
      • Decant cleared lysate into Spin Column
      • Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough, and reinsert Column into Collection Tube.
    • Washing
      • Add 750 µl of Wash Solution (ethanol added). Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into Collection Tube
      • Repeat step before with 250 µl of Wash solution
      • Centrifuge at top speed for 2 minutes at room temperature
    • Elution
      • Transfer Spin Column to a sterile 1.5 ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column. If the Spin Column has Column Wash Solution associated with it, centrifuge again for 1 minute at top speed, then transfer the Spin Column to a new, sterile 1.5 ml microcentrifuge tube
      • Add 100 µl of Nuclease-Free Water to the Spin Column. Centrifuge at top speed for 1 minute at room temperature
      • Discard column, and store DNA at -20°C or below

  • GeneJET Plasmid Miniprep Kit by Thermo Scientific
    • Note:
      • All purification steps should be carried out at room temperature.
      • All centrifugation should be carried out in a table-top microcentrifuge at >12000 x g
        (10 000-14 000 rpm, depending on the rotor type).

    • Resuspend the pelleted cells in 250 µl of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
      • Note Ensure RNase A has been added to the Resuspension Solution.
    • Add 250 µl of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
      • Note Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 minutes to avoid denaturation of supercoiled plasmid DNA.
    • Add 350 µl of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
      • Note It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudly.
    • Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA.
    • Transfer the supernatant to the supplied GeneJET spin coloumn by decanting or pipetting. Avoid disburbing or transferring the white precipitate.
    • Centrifuge for 1 minute. Discard the flow-through and place the coloumn back into the same collection tube.
      • Note Do not add bleach to the flow-through.
    • Add 500 µl of the Wash Solution (diluted with ethanol prior to first use) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
    • Repeat the wash procedure (step before) using 500 µl of the Wash Solution.
    • Discard the flow-through and centrifuge for an additional 1 minute to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
    • Transfer the GeneJET spin column into a fresh 1.5 ml microcentrifuge tube (not included). Add 50 µl of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmide DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 minutes at room temperature and centrifuge for 2 minutes.
      • Note An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
    • Discard the column and store the purified plasmid DNA at -20°C.

  • QIAprep® Spin Miniprep Kit by QIAGEN
    • Note: All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional able-top microcentrifuge.

    • Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15-25 °C).
    • Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
    • Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
    • Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless.
    • Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
    • Apply the supernatant from step 5 to the QIprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source.
    • Recommended: Wash the QIAprep spin column by adding 500 µl Buffer PB. Centrifuge for 30-60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIprep spin column and switch off the vacuum source.
    • Wash the QIAprep spin column by adding 750 µl Buffer PE. Centrifuge for 30-60s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source. Transfer the QIAprep spin column to the collection tube.
    • Centrifuge for 1 min to remove residual wash buffer
    • Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris*Cl, pH 8.5) or water to the center of the QIAprep spin column. Let it stand for 1 min, and centrifuge for 1 min.
Generating chemocompetent cells

Generating chemocompetent cells

  • Material:
    • 300 ml LB-Medium
    • ~10 ml cooled TSS buffer
    • 5 pre-cooled 50 ml Falcons
  • Protocol:
    • Inoculate 2x3 ml LB with bacterial stock; incubate over night at 37 °C and 200 rpm
    • Inoculate 1x250 ml LB with the over night cultures in 1 ml flask at 37 °C and 140 rpm
    • Incubate until OD600 0.4-0.6
    • Devide the culture into five cooled 50 ml Falcons and incubate on ice for 10 minutes
    • Onwards all steps at 4°C
    • Centrifugate at 3000 rpm, 4 °C for 10 minutes
    • Discard supernatant
    • Pipette out remaining media
    • The volume of TSS to use is 10% of the culture volume that you spun down (we only used 1-2 ml). You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
    • Use 100 µl per aliquot and drop them in liquid nitrogen immediatly
    • Store at -80 °C
Generating electrocompetent cells

Generating electrocompetent cells

  • Material:
    • 550 ml LB-Medium
    • 1 L cooled bidest. H2O
    • 50 ml cooled 10 % glycerine
    • 10 pre-cooled 50 ml Falcons
  • Protocol:
    • Inoculate 2x3 ml LB with bacterial stock; incubate over night at 37 °C and 200 rpm
    • Inoculate 2x250 ml LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm
    • Incubate until OD600 0.4-0.6
    • Cool the culture 15-30 minutes on ice
    • Onwards all steps at 4°C
    • Divide the cultures into cooled 50 ml Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate
    • Discard supernatant
    • Resuspend pellet in 5 ml cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)
    • Pool two suspensions each, add bidest H2O up to 50 ml and centrifugate again (see centrifugation above)
    • Discard supernatant
    • Resuspend pellet in 5 ml cooled bidest H2O
    • Add bidest H2O up to 50 ml and centrifugate again (see centrifugation above)
    • Discard supernatant
    • Resuspend pellet in 5 ml cooled 10 % glycerine
    • Transfer suspensions in two 50 ml Falcons and centrifugate again (see centrifugation above)
    • Discard supernatant
    • Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend
    • Divide cells in 50 µl aliquots and freeze in liquid N2 immediately
    • Store at -80 °C
Gibson Assembly

Gibson Assembly
  • Modified from Gibson et al. (2009)
  • This assembly method is an isothermal, single-reaction method for assembling multiple overlapping DNA molecules. By coordinating the activity of a 5‘ exonuclease, a DNA polymerase and a DNA ligase two adjacent DNA fragments with complementary terminal sequence overlaps can be joined into a covalently sealed molecule, without the use of any restriction endonuclease.


    • Gibson-Assembly
  • Preparation of DNA molecules for in vitro recombination
    • Generate the complementary sequence overlaps by PCR using the Phusion DNA-polymerase. If necessary add 5 M Betain in the reaction mix by reducing the amount of H2O to decrease the number of false PCR products.
    • Identify the PCR products of interest by gel electrophoresis with known DNA standards.
    • Extract the PCR products from the gel by cutting out the DNA fragments and clean them up by using a commercial PCR clean-up kit.
  • in vitro recombination
    • Assembly mixture:
      • 320 µl 5x isothermal reaction buffer
      • 0.64 µl of 10 U ml-1 T5 exonuclease (for DNA molecules overlapping by greater than 150 bp add 3.2 µl of 10 U ml–1 T5 exonuclease)
      • 20 µl of 2 U ml-1 Phusion DNA polymerase
      • 160 µl of 40 U ml-1 taq DNA ligase
      • add ddH2O water up to a final volume of 1.2 ml
      • aliquote 15 µl of the reagent-enzyme mix and store it at –20 ˚C
    • Thaw 15 µl assembly mixture aliquot and keep it on ice until use.
    • Add 5 µl of the purified DNA molecules in equimolar amounts (between 10 and 100 ng of each DNA fragment).
    • Incubate the resulting mixture at 50 ˚C for 15 to 60 min, with 60 min being optimal.
    • Transformation ( via heat shock or via electroporation) without cleaning up the assembly product.
NEB BioBrick Assembly Kit   (NEB)

NEB BioBrick Assembly Kit   (NEB)

Gibson-Assembly
  • We used the BioBrick Assembly Kit to assemble an upstream part with a downstream part into destination plasmid.
  • Digestion Protocoll:
    • Digest upstream part with EcoRI-HF and SpeI
    • Digest downstream part with XbaI and PstI
    • Digest destiantion destination plasmid with EcoRI-HF and PstI
      • 500 ng part DNA
      • 1 μL of each enzyme
      • 5 μL 10x NEBuffer 2.1
      • to 50 μL H20
    • Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.
  • Dephosphorylation Protocoll
    • Add 1 μL of AP (Antarctic phosphatase) and 5 µl of 10x AP reaction buffer to digested destination plasmid, incubate for 1 h at 37 °C.
  • Ligation Protocoll
    • Ligate the upstream and downstream parts into the digested destination plasmid.
      • 2 μL of each part
      • 2 μL 10x T4 DNA Ligase Buffer
      • 1 μL T4 DNA Ligase
      • 11 μL H2O
    • Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.
  • Transform 1-2 μL of the ligation product into 50 μL of competent E. coli cells.
Standard BioBrick Assembly

Standard BioBrick Assembly
  • modified from silver lab
  • This assembly method can be used for BioBricks which are bigger than 150 bp. The BioBrick should be at least 500 bp bigger or smaller than the backbone. The BioBrick, which complies with these conditions, is used as the insert and is assembled into the prefix or suffix of the other used BioBrick, called vector. So you have to differentiate between a prefix and a suffix insertion.

    • Silver Suffix Insertion

    Silver Prefix Insertion
  • Suffix insertion:
    • Digestion of insert: at least 700 ng DNA / 10 µl volume, 1 µl 10x NEBuffer 2.1, 0.5 µl XbaI, 0.5 µl PstI. Digest for 1 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
    • Digestion of vector about 700 ng DNA / 10 µl volume, 1 µl 10x NEBuffer 2.1, 0.5 µl SpeI, 0.5 µl PstI. Digest for 1 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µl AP (Antarctic phosphatase) and 1.2 µl 10 x AP reaction buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit.
    • Ligation: after digestion and clean-up: 50 - 200 ng of vector, 3 - 10 fold molar access of insert, 20 µl ligation volume, 2 µl T4-Ligase-Buffer, 1 µl T4-Ligase. Incubate for 20 - 30 min at room temperature, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.
  • Prefix insertion:
    • Digestion of insert: at least 700 ng DNA / 10 µl volume, 1 µl 10x NEBuffer 2.1, 0.5 µl EcoRI, 0.5 µl SpeI. Digest for 1 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
    • Digestion of vector about 700 ng DNA / 10 µl volume, 1 µl 10 x NEBuffer 2.1, 0.5 µl EcoRI, 0.5 µl XbaI. Digest for 1h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µl AP (Antarctic phosphatase) and 1.2 µl 10 x AP reaction buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit.
    • Ligation: after digestion and clean-up: 50 - 200 ng of vector, 3 - 10 fold molar access of insert, 20 µl ligation volume, 2 µl T4-Ligase-Buffer, 1 µl T4-Ligase. Incubate for 20 - 30 min at room temperature, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.
  • Variations:
    • A digestion over night is possible. If you digest over night use only 0.1 µl restriction enzyme.
    • It is also possible to use PCR product as insert. Digest after PCR with corresponding restriction enzymes and clean up with a PCR clean-up kit. This could lead to higher yields of insert DNA because a lot of DNA gets lost during the gel electrophoresis clean up.
    • Sometimes some BioBricks are hard to assemble. Then you have to clean up the vector by gel electrophoresis as well.
Transformation via electroporation

Transformation via electroporation

  • Thaw 50 µl electrocompetent E. coli cells on ice, dilute with icecold 50 µl glycerine (10 %) if necessary
  • Add 0.5-5 µl plasmid to 50 µl electrocompetent cells
  • Store cells on ice for 1 minute
  • Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ω
  • Transfer transformation reaction to 450 µl SOC-Medium and incubate 1 h at 37 °C
  • Centrifuge 3 minutes at 1200 rpm and plate on selective LB-Medium
  • Incubate over night at 37 °C
Transformation via heat shock

Transformation via heat shock

  • Thaw 100 µl chemocompetent E. coli cells on ice
  • Add 0.5-5 µl plasmid to 100 µl chemocompetent cells
  • Store cells on ice for 10-30 minutes
  • Heat shock for 90 seconds at 42 °C
  • Transfer transformation reaction to 450 µl SOC-Medium and incubate 1 h at 37 °C
  • Centrifuge 3 minutes at 1200 rpm and plate on selective LB-Medium
  • Incubate over night at 37 °C
Analytics
Cultivations

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