Team:Bielefeld-Germany/Protocols

From 2011.igem.org

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===SDS-PAGE gel===
===SDS-PAGE gel===
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The amouts in following the tabe for one gel.
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The amouts in following tabe are for one gel.
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Revision as of 14:36, 28 August 2011

Contents

Transformation via electroporation

  • Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerol (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 = 200 Ώ
  • Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
  • Centrifuge 2 min at 800 rpm and plate on selective LB-Medium

Transformation of Single Step (KRX) Competent Cells by Promega

using protocol for E. coli KRX single step competent cells by Promega

  • Remove Single Step (KRX) Competent Cells from –70 °C, and place on ice for 5 minutes or until just thawed.
  • Add 1–50 ng of DNA (in a volume not greater than 5 μL) to the Single Step (KRX) Competent Cells. Move the pipette tip through the cells while dispensing. Quickly flick the tube several times. Do not vortex!
  • Immediately return the tubes to ice for 5–30 minutes
  • Heat-shock cells for 15–20 seconds in a water bath at exactly 42 °C. Do not shake.
  • Immediately place the tubes on ice for 2 minutes.
  • Add 450 μL of room-temperature SOC medium to each transformation reaction, and incubate for 60 minutes at 37 °C with shaking (approximately 225 rpm). For best transformation efficiency, lay the tubes on their sides and tape them to the platform.
  • For each transformation reaction, we recommend plating 100 μL of undiluted cells and 1:10 and 1:100 cell dilutions on antibiotic plates. Incubate the plates at 37 °C overnight.

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 Tango buffer, 0.5 µL XbaI, 1 µL PstI. Digest for 2 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 orange buffer, 0.5 µL SpeI, 0.5 µL PstI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP 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 BamHI buffer, 0.5 µL EcoRI, 0.5 µL SpeI. Digest for 2 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 Tango buffer, 0.5 µL EcoRI, 0.5 µL XbaI. Digest for 2h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP 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 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.


Standard Freiburg BioBrick Assembly

modified from Silver lab and Assembly standard 25:

This assembly method can be used for fusion protein assemblies with 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, which is called vector and needs to be available in the BioBrick Assembly standard 25. You have to differentiate between a prefix and a suffix insertion.

Suffix Insertion

  • Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x NEB buffer 4 + 0.1 µL 100x BSA, 0.5 µL NgoMIV (NEB), 1 µL PstI. Digest for 2 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 orange buffer, 0.5 µL AgeI, 0.5 µL PstI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10x SAP 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 orange buffer, 0.5 µL EcoRI, 0.5 µL AgeI. Digest for 2 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.
  • 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 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.


Standard 3A assembly

Modified from BioBrick Assembly Manual by Ginkgo BioWorks

Digestion

  • Thaw DNA from upstream and downstream part and the destination plasmid on ice.
    • Destination plasmid has to carry the ccdB gene <partinfo>P1010</partinfo> as insert and has to have a different antibiotic resistance than the plasmids carrying the upstream and downstream parts
    • DNA has to be cleaned (by MiniPrep or after a PCR)
  • 500 ng DNA / digestion mix for upstream part, downstream part and destination plasmid (total volume of mix 10 µL, dilute with ddH20 if necessary)
Upstream part Downstream part Destination plasmid
enzyme 1 0.5 µL EcoRI 0.5 µL XbaI 0.5 µL EcoRI
enzyme 2 0.5 µL SpeI 1 µL PstI 0.5 µL PstI
buffer BamHI Tango Orange


  • Incubation of the digestion mixes at 37 °C
  • After another hour: heat inactivation of all mixes for 20 min at 80 °C
  • Continue with ligation or freeze the mixes

Ligation

  • Ligation mix:
    • 2 µL ddH2O
    • 5 µL of every digestion mix (so 15 µL in total)
    • 2 µL T4-DNA-ligase buffer (thaw on ice!)
    • 1 µL T4-DNA-ligase
  • Incubate at least 20 min at room temperature, afterwards heat inactivation for 5 min at 70 °C (optional)
  • Freeze ligation mix or continue with transformation (heatshock or electroporation)


Freiburg 3A assembly

Modified from BioBrick Assembly Manual by Ginkgo BioWorks and Assembly standard 25

Digestion

  • Thaw DNA from upstream and downstream part (=N-terminal and C-terminal protein domain) and the destination plasmid on ice.
    • Destination plasmid has to carry the ccdB gene <partinfo>P1010</partinfo> as insert and has to have a different antibiotic resistance than the plasmids carrying the upstream and downstream parts
    • DNA has to be cleaned (by MiniPrep or after a PCR)
  • 500 ng DNA / digestion mix for upstream part, downstream part and destination plasmid (total volume of mix 10 µL, dilute with ddH20 if necessary)
Upstream part Downstream part Destination plasmid
enzyme 1 0.5 µL EcoRI 0.5 µL NgoMIV 0.5 µL EcoRI
enzyme 2 0.5 µL AgeI 1 µL PstI 0.5 µL PstI
buffer Orange NEB buffer 4 + BSA Orange


  • Incubation of the digestion mixes at 37 °C
  • After another hour: heat inactivation of all mixes for 20 min at 80 °C
  • Continue with ligation or freeze the mixes

Ligation

  • Ligation mix:
    • 2 µL ddH2O
    • 5 µL of every digestion mix (so 15 µL in total)
    • 2 µL T4-DNA-ligase buffer (thaw on ice!)
    • 1 µL T4-DNA-ligase
  • Incubate at least 20 min at room temperature, afterwards heat inactivation for 5 min at 70 °C (optional)
  • Freeze ligation mix or continue with transformation (heatshock or electroporation)

Gibson assembly

Gibson Assembly

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.

Preparation of DNA molecules for in vitro recombination

  • Generate the complementary sequence overlaps by Phusion-PCR. If necessary use ??? M Betain in the reaction mix by reducing the amount of H2O to decrease the number of wrong PCR products.
  • Identify the PCR pruducts of interest by gel electrophoresis with known DNA standards.
  • Extract the PCR products from the gel by cutting the DNA fragment out and clean them up by using a commercial 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 (heatshock or electroporation) without cleaning up the assembly product.

Restriction analysis

  • Digest BioBrick of interest: about 400 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL NotI or PstI. Digest for 2 h at 37 °C. NotI is used to determine the length of the BioBrick and the plasmid backbone, PstI ist used to determine the length of the BioBrick in the plasmid backbone.
  • Gel electrophoresis: add 2 µL loading buffer to every digestion mix, apply about 100 - 200 ng DNA / pocket in gel. Don't forget to apply the uncut BioBrick as well. A good agarose concentration for BioBricks between 0.2 and 3 kb is 1.5 %. The smaller your BioBrick of interest is the higher the agarose concentration should be and vice versa. The gel electrophoresis is made with TAE-buffer. Be sure that you melt your agarose gel in the same buffer you use for the electrophoresis later.

Colony PCR

  • Pick one colony with a sterile tip and elute it in 100 µL ddH20 or medium
  • Store the colony in 4 °C while colony PCR is running
  • One reaction mix contains:
    • 10 µL 5x buffer
    • 2 µL MgCl2 (25 mM stock)
    • 1 µL dNTPs
    • 0.5 µL primer mix (prefix/suffix primers or sequencing primers)
    • 35.25 µL ddH2O
    • 0.25 µL GoTaq polymerase (Promega)
    • 1 µL template
  • PCR program:
    • Start: 3 min, 98 °C
    • 30 cycles of:
      • 30 s, 98 °C
      • 30 s, 55 °C
      • 30 s / 1 kb template, 72 °C
    • Finish: 5 min, 72 °C
  • Gel electrophoresis: check the fragment size
  • Plate the correct colony

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

This analytical Method can be used for separation and identification of proteins according to their electrophoretic mobility. The mobility is a function of length of the molecular weight. Proteins that have identical charge per unit mass due to binding of SDS results in an equal electrophoretic mobility.

Pouring the polyacrylamide gel

  • Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED.
  • Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel.
  • Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel.
  • layer isopropanol on top of the gel.
  • Leave the separating gel at room temperature for >60 minutes to polymerize.
  • Remove isopropanol and wait until the surface is dry.
  • Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form.
  • Insert comb without getting bubbles stuck underneath
  • Leave the gel at room temperature for >60 minutes to polymerize.
  • For storage
    • Remove sealing and store the gel wrapped in moistened paper towel at 4°C.

Preparing the sample

  • Mix you protein 4:1 with Laemmli-buffer (30 mL proteinsolution + 10 mL Laemmli-buffer)
  • heat for 5 minutes at 95 °C.

Running the gel

  • Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber.
  • Remove comp without destroying the gel pocket.
  • Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample.
  • Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel.
  • Raise amperage up to 20 mA for running the separating gel.
  • When the distance of the lowest molecular weight standard lane to the gel end is down to 0,5 cm stop the electrophoresis by turning off the power supply.

Bisphenol A analysis

Extraction with ethylacetate

  • mix 100 µL culture supernatant with 100 µL internal standard ( bisphenol F, 100 µg L-1)
  • add 200 µL ethylacetate (HPLC grade) for extraction
  • vortex (30 s)
  • centrifuge for phase separation (5 min, 5000 g)
  • take a bit from upper phase and put it in a clean eppi
  • SpeedVac at 40 °C to remove ethlyacetate
  • solve remaining BPA in water (HPLC grade), vortex (30 s)
  • solubility of BPA in water only 300 mg L-1
    • for LC-MS analysis of BPA, 300 mg BPA L-1 is definitely enough
    • if you want to detect or expect higher concentrations of BPA, solve it in an acetonitrile-water-mix

HPLC method

  • C18 reverse phase column
  • gradient starting with 45 % acetonitrile up to 95 % acetonitrile

NAD+ detection

For the design of Molecular Beacons different available tools may help you so that a correct functionality is ensured under defined assay conditions. All measurements were made in the NAD+ bioassay buffer.

Characterisation of Molecular Beacons

Preferentially, use a complementary oligonucleotide (target) as well as two enshortened oligonucleotides (split target) for characterising the Molecular Beacon in its closed and open state. In this way you can verify simultaneously the ineffectiveness of the split target to reach the Molecular Beacon`s open state.

  • Optimal wavelengths:
    • Determine the emission spectrum of your Molecular Beacon by initially using a literature value for the exctintion wavelength of the labeled fluorophore. For this add 10 µL of 1 µM Molecular Beacon in 200 µL NAD+ bioassay buffer, mix thoroughly and wait 2 min until measuring the fluorescence with a spectrofluorometer at an defined temperature (optimally 37 °C) .
    • Add 10 µL of 2 µM either target or split target and shake at least 6 min until measuring the fluorescence again (when equilibrium is reached).
    • Repeat the procedure to determine the extinction spectrum using the self-calculated optimal emission wavelength for your fluorophore/quencher combination. For further fluorescence measurements use the extinction wavelength for which the difference between the Molecular Beacons's closed state and the open state signals is maximal.
  • Thermal profile analysis:
    • Prepare a 30 µL reaction mix composed of NAD+ bioassay buffer and 1 µM Molecular Beacon. In a separate sample add 2 µM either target or split target.
    • Use a spectrofluorometric thermal cylcer to dertemine the fluorescence as a function of temperature. Start at 80 °C and decrease the temperature stepwise in 1 °C intervals holding each temperature for 1 min and measuring the fluorescence at the end of each step.
  • Signal-to-background ratio (S/B):
Signal-to-background ratio
    • The approach is quite similar to the one that determines the optimal wavelengths which should be used to monitor the fluorescence of the NAD+ bioassay buffer (Fbuffer) and the added Molecular Beacon (Fclosed) as well as the target (Fopen) or split target. Monitor the fluorescence until the equilibrium is reached each time before you add a new component.
    • The signal-to-background ratio can be calculated as shown on the right. Finally, compare the signal-to-background ratios after adding the target and split target to check whether the split target has an effect on the melting of the Molecular Beacon's hairpin structure.
  • Imaging:
    • Prepare a 200 µL reaction mix composed of NAD+ bioassay buffer and 500 nM Molecular Beacon in a PCR tube. In a separate sample add 600 µM either target or split target. Excitate the samples by a UV transilluminator and take images preferably with a camera that can detect different colours.

Purification of DNA Ligase

NAD+ bioassay

Used enzymes

Enzyme Producer
AgeI Fermentas
DpnI Fermentas
EcoRI Fermentas
GoTaq DNA-polymerase Promega
KOD Hotstart DNA-polymerase Novagen
NgoMIV NEB
OneTaq DNA-polymerase NEB
Pfu DNA-polymerase Promega
PstI Fermentas
Phusion HF DNA-polymerase Finnzymes
Shrimp alcaline phosphatase Fermentas
SpeI Fermentas
T4-DNA-Ligase Fermentas
taq DNA-polymerase Bioline

Used Kits

Function Name
Molecular Cloning Fermentas CloneJET™ PCR Cloning Kit
Plasmid purification Fermentas GeneJET™ Plasmid Miniprep Kit
Plasmid purification Promega PureYield™ Plasmid Preps
PCR Cleanup Macherey Nagel NucleoSpin® Extract II
PCR Cleanup Promega Wizard® SV Gel and PCR Clean-Up
PCR core system Promega GoTaq® PCR Core System I

Media, buffer, solutions etc.

TAE buffer

For 1 L of 50 x TAE buffer you need:

  • 242.48 g Tris
  • 41.02 g Sodiumacetate
  • 18.612 g EDTA
  • Adjust pH to 7.8 with acetic acid
  • Solve in dH2O

10 mL of the stock is diluted in 1 L dH2O for the gel electrophoresis (0.5 x TAE buffer).


DNA loading buffer

  • 50 % (v/v) glycerol
  • 1 mM EDTA
  • 0.1 % (w/v) bromphenol blue
  • Solve in ddH2O


LB medium

For 1 L of LB medium you need:

  • 10 g Trypton
  • 5 g yeast extract
  • 10 g NaCl
  • 12 g Agar-Agar (for plates)
  • Adjust pH to 7.4


M9 medium

For 250 mL M9 medium you need 175 mL sterile water (for plates add 4 g Agar-Agar as well). Then add (in the following order:

  • 250 µL 100 mM CaCl2
  • 2.5 mL trace salts
    • store this stock solution in the dark
  • 250 µL MgSO4
  • 250 µL 50 mM FeCl3 / 100 mM citrate (one solution, citrate is iron carrier)
    • store this stock solution cold and in the dark
  • carbon source stock solution (e.g. glucose)
  • 50 mL 5x M9 salts stock solution
    • 64 g L-1 Na2HPO4 * 7 H2O
    • 15 g L-1 KH2PO4
    • 2.5 g L-1 NaCl
    • 5 g L-1 NH4Cl
  • antibiotic stock solution
  • fill up to 250 mL with sterile water


5x isothermal reaction buffer for Gibson assembly

storage -20˚C

  • 3 mL of 1 M Tris-HCl (pH 7.5)
  • 150 µL of 2 M MgCl2,
  • 60 µL of 100 mM dGTP
  • 60 µL of 100 mM dATP
  • 60 µL of 100 mM dTTP,
  • 60 µL of 100 mM dCTP
  • 300 µL of 1 M DTT
  • 1.5 g PEG-8000 and
  • 300 µL of 100 mM NAD

SDS-PAGE gel

The amouts in following tabe are for one gel.

Type of gel Component Volume
stacking gel 5 % H2O 775 μL
0,25 M Tris (pH 6,8) 1,25 mL
Bis/Acrylamide (0,8 %, 30 %) 425 μL
5 % SDS 50 μL
10 % Ammonium persulfate 25 μL
TEMED 3 μL
separating gel 12 % H2O 1,5 mL
1 M Tris (pH 8,8) 2,8 mL
Bis/ Acrylamide (0,8%, 30%) 3,0 mL
5% SDS 150 μL
10% Ammonium persulfate 37,5 μL
TEMED 5 μL

NAD+ bioassay buffer

  • 50 mM Tris-HCl, pH 8.0
  • 10 mM MgCl2
  • 2,5 mM CaCl2
  • 5 mM DTT
  • 0,05 % BSA

Used chemicals

Chemical Producer Purity
Acetonitrile VWR 99.9 %, HPLC Grade
Bisphenol F Alfa Aesar 98 %
Ethylacetate VWR > 99.5 %, p.a.
Chemical A Producer A XX.X %
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