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Genetic engineering

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

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

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)

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 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

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

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

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 nitrogen immediately
Store at -80 °C

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)

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

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

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

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

Sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE)

Pouring the polyacrylamide gel:

For each separating gel (12 %) aliquote:

1.35 mL Bisacrylamid/Acrylamid (0.8 % , 30 %, at the ratio of 37.5:1)
0.675 mL H₂O
0.675 mL 1.88 M Tris-HCl (pH 8.8)
0.675 mL 0.5 % SDS

Add 120 µl 10 % ammonium persulfate and 37,5 µl TEMED to each aliquote and mix
Pour the solution quickly into the 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 wash the surface of the separating gel with H₂O. Wait until the surface is dry
For each stacking gel (5 %) aliquote:

0.309 mL Bisacrylamid/Acrylamid (0.8 % , 30 %, at the ratio of 37.5:1)
0.726 mL H₂O
0.375 mL 0.625 M Tris-HCl (pH 6.8)
0.375 mL 0.5 % SDS

Add 60 µl 10 % ammonium persulfate and 22,5 µl TEMED to each aliquote and mix
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 your protein mixture 4:1 with PBJR buffer (15 µL protein solution + 5 µL PBJR buffer)
Heat for 5 minutes at 95 °C

Running the gel:

Remove sealing, put the polymerized gel into gel box and pour SDS-PAGE running buffer into the negative and positive electrode chamber
Remove comp without destroying the gel pockets
Pipet the SDS running buffer in the gel pockets up and down for flushing the gel pockets
Pipet slowly 20 µL of the sample into the gel pockets
Make sure to include at least one lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of the 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

Staining the polyacrylamide gel (Colloidal Coomassie Brilliant Blue staining):

After finishing the SDS-PAGE remove gel from gel casting form and transfer it into a box
Add 100 mL of the Colloidal Coomaassie Brilliant Blue staining solution to your polyacrylamid gel
Incubate the gel in the solution at room temperature until the protein bands got an intensive blue color. Shake the gel continuously during incubation
Remove the staining solution
Wash the gel with 7 % (v/v) acetic acid in H₂O for decoloration
Incubate the gel in H₂O (2-6 h) for bleaching the background. Shake the gel continuously during incubation. If necessary replace the colored water with new one

Matrix-assisted Laser Desorption/Ionization – Time of flight (MALDI-TOF)

Tryptic digest of gel lanes for analysis with MALDI-TOF:

Make sure to work under a fume hood
Do not work with protective gloves to prevent contamination of your sample with plasticizers
Reaction tubes have to be cleaned with 60 % (v/v) CH₃CN and 0.1 % (v/v) TFA. Afterwards the solution has to be removed completely followed by evaporation of the tubes under a fume hood
Cut out the protein lanes of a Coomassie-stained SDS-PAGE using a clean scalpel. Gel parts are transferred to the washed reaction tubes. If necessary cut the parts to smaller slices
Gel slices should be washed two times. Therefore add 200 µL 30 % (v/v) acetonitrile in 0,1 M ammonium hydrogen carbonate each time and shake lightly for 10 minutes. Remove supernatant and discard to special waste
Dry gel slices at least 30 minutes in a Speedvac
Rehydrate gel slices in 15 µL Trypsin-solution followed by short centrifugation

Trypsin-solution: 1 µL Trypsin + 14 µL 10 mM NH₄HCO₃

For this solution solubilize lyophilized Trypsin in 200 µL of provided buffer and activate Trypsin for 15 minutes at 30 °C. For further use it can be stored at -20 °C

Preparation and Spotting for analysis of peptides on Bruker AnchorChips:

Spot 0,5 - 1 µL of sample aliquot
Add 1 µL HCCA matrix solution to the spotted sample aliquots. Pipet up and down approximately five times to obtain a sufficient mixing. Be careful not to contact the AnchorChip. Note: Most of the sample solvent needs to be gone in order to achieve a sufficiently low water content. When the matrix solution is added to the previously spotted sample aliquot at a too high water content in the mixture, it will result in undesired crystallization of the matrix outside the anchor spot area
Dry the prepared spots at room temperature
Spot external calibrants on the adjacent calibrant spot positions. Use the calibrant stock solution (Bruker’s “Peptide Calibration Standard II”, Part number #222570), add 125 µL of 0.1 % TFA (v/v) in 30 % ACN to the vial. Vortex and sonicate the vial
Mix the calibrant stock solution in a 1:200 ratio with HCCA matrix and deposit 1 µL of the mixture onto the calibrant spots
Analyze samples in ultrafleXtreme by Bruker Daltonics

Fast Cell Lysis for SDS-PAGE

Measure OD₆₀₀
Transfer two samples a 1,5 ml in a microcentrifuge tube
Centrifuge at top speed for 5 minutes at room temperature
Discard supernatant quantitative
Store pellet at -20 °C
Thaw pellet and resupend in Fast Cell Lysis sample buffer. (30 µL Fast Cell Lysis sample buffer. per OD₆₀₀)
Heat for 5 - 10 minutes at 95 - 100 °C
Centrifuge at top speed for 15 minutes at room temperature
Transfer supernatant to a new microcentrifuge tube
Analyze samples by SDS-PAGE. Use 3 - 10 µL per sample

Amicon Ultra - Protein buffer exchange and desalting

Load up to 15ml of purified enzyme on to the Amicon Ultra filter device
Place capped filter device into centrifuge rotor; counterbalance with a similar device
When using a swinging bucket rotor, spin the device at 4,000xg maximum for approximately 15-60 minutes
When using a fixed angle rotor, orient the device with the membrane panel facing up and spin at 5,000xg maximum for approximately 15-60 minutes
To recover the the concentrated solute, insert a pipettor into the bottom of the filter device and withdraw the sample using a side-to-side sweeping motion to ensure total recovery. The ultrafiltrate can be stored in the centrifuge tube.
For buffer exchange load up to 15ml of new buffer on to the Amicon Ultra filter device and centrifuge for 15-60 minutes

His Trap FF Purification

Column preparation

1 h Incubation with 1 M NaOH
Wash with 10 ml Binding Buffer
Wash with 10 ml distilled water
Strip the column with 10 ml Stripping Buffer
Wash with 10 ml Binding Buffer
Wash with 10 ml distilled water
Recharge with 2.5 ml 0.1 M NiSO₄
Wash with 5 ml distilled water
Wash with 5ml Binding Buffer
Perform a blank run:

Wash with 5 ml distilled water (After Ethanol storage)
Wash with 5 ml Binding Buffer
Wash with 5 ml Elution Buffer 5
Equilibrate with 10ml Binding Buffer

Store in 20% ethanol

Sample preparation:

Add 5 ml Binding Buffer to each gram of pellet
Add 0.2 mg/ml lysozyme, 20 µg/ml DNAse, 1 mM MgCl₂, 1 mM PMSF
Stir for 30 min at +4°C up to +20°C (depending on the protein)
Centrifuge 30 min at 4°C
Adjust pH to 7.4 - 7-6
Apply the lysate on the column immediatly

Sample run:

Wash with 5 ml distilled water
Equilibrate with 5 ml Binding Buffer
Apply the lysate on the column
Wash with Binding Buffer until the absorbance reaches a steady baseline (10 ml)
Elute with 5 ml Elution Buffer gradient (Elution Buffer 1 – 5)

Cultivations

Protein Expression under control of the T7 promotor

This protocol is based on the technical bulletin from Promega

Grow overnight culture of your cells with the plasmid containing the T7 promotor in LB medium containing specific antibiotic. Grow at 37 °C
Dilute overnight culture 1:50 in LB medium containing antibiotic and grow cells at 37 °C
When the culture reaches an OD₆₀₀ of 0.4 - 0.5, shift the temperature to 20 °C
When an OD₆₀₀ of 0.6-0.8 is reached, induce protein expression. Add rhamnose to a final concentration of 0,1 %
Grow the culture at 20 °C overnight
Harvest cells by centrifugation at 4 °C
Discard supernatant and freeze pellet at -20 °C

@@ Line 834: / Line 834: @@
 <li> Discard supernatant quantitative </li>
 <li> Store pellet at -20 °C </li>
-<li> Thaw pellet and resupend in sample buffer (30 µL sample buffer per OD<sub>600</sub>) </li>
+<li> Thaw pellet and resupend in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#Fast%20Cell%20Lysis%20sample%20buffer" target="_blank">Fast Cell Lysis sample buffer.</a> (30 µL <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#Fast%20Cell%20Lysis%20sample%20buffer" target="_blank">Fast Cell Lysis sample buffer.</a> per OD<sub>600</sub>) </li>
 <li> Heat for 5 - 10 minutes at 95 - 100 °C </li>
 <li> Centrifuge at top speed for 15 minutes at room temperature </li>

Team:Bielefeld-CeBiTec/Notebook/Protocols

From 2014.igem.org

Revision as of 21:17, 9 October 2014

Protocols

PCR

Colony PCR

DNA isolation (genomic DNA)

DNA Purification by Centrifugation

Plasmid isolation

Generating chemocompetent cells

Generating electrocompetent cells

Gibson Assembly

NEB BioBrick Assembly Kit (NEB)

Standard BioBrick Assembly

Transformation via electroporation

Transformation via heat shock

Sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE)

Matrix-assisted Laser Desorption/Ionization – Time of flight (MALDI-TOF)

Fast Cell Lysis for SDS-PAGE

Amicon Ultra - Protein buffer exchange and desalting

His Trap FF Purification

Protein Expression under control of the T7 promotor