Agarose Gel Electrophoresis

Prepare the Agarose Gel:

1. Weigh appropriate amount of agarose into a small Erlenmeyer flask (0.3g for 30mL small 1% gel).
2. Add desired volume of 1x TAE buffer (or 1x TBE in special cases) (30mL for small gel chamber).
3. Weight the flask and write down its total weight.
4. Boil it to resolve the agarose:
   • either by microwaving (without magnetic stir bar)
   • or on hot plate (with magnetic stir bar).
5. Let it cool down while stirring to about 60°C (hand-warm).
6. Weight again and replace lost water.
7. Seal ends of gel plate with black wedges.
8. Cast gel into gel plate and put in the comb, wait until solid.

Prepare Samples:

1. Transfer about 0.3μg DNA into a microcentrifuge tube.
2. Add 1x TAE to 5μL.
3. Add 1μL of 6x DNA sample buffer.

Run Electrophoresis:

1. Place gel into the big chamber in correct orientation (DNA migrates to the positive pole)
2. Fill chamber with 1x TAE buffer (or 1x TBE), about 0.5cm above gel.
3. Carefully remove comb from solid gel.
4. Load the 6 μL samples into the gel slots, and load 4 μL of DNA ladder (typically 1kb ladder) in at least one gel slot.
5. Close chamber with lid in the correct orientation (black/red).
6. Connect cables to power supply.
7. Run the gel with 1–5V/cm of gel length, check for dye fronts should be at about 1/3 and 2/3 of gels (about 1h) – common lab procedure: 100V for small gel.

Staining and Photo:

1. Stain the gels for 10–20 minutes in ethidiumbromide while shaking. Attention: always wear gloves with ethidiumbromide, try not to touch the solution or the stained gel but use a kitchen spatula to handle the gel.
2. Destain the gel for a short while in water (e.g. while transporting it to take the photo).
3. Observe bands on UV illuminator and take a digital photo. Carfeful: strong UV light, wear glasses and lab coat to avoid “sun burn."
4. Clean UV illuminator.
5. Discard gel in ethidiumbromide waste.

Buffers:

Gene Ruler 1kb DNA ladder (Fermentas)

Results in 0.08333μg/μL, i.e. 0.5μg in 6μL as recommended by Fermentas

Gel Concentration to Separate Various Sizes of DNA:

* For gels of more than 2%, use 3 parts “cheap” agarose and 1 part Top Vision Agarose, e.g. 0.9g cheap agarose + 0.3g Top Vision (not low melting) agarose for 40mL of 3.% gel

Overnight Cultures

Overnight Culture of Bacteria for Plasmid Purification:

1. Using proper Aseptic technique, sterilely transfer 5mL of LB medium into a sterile culture tube or falcon tube.
2. Thaw the appropriate antibiotic and pipette in the amount needed.
3. Sterilize the inoculating loop using flame and allow cooling then carefully picking one colony from a plate or scraping some media from a glycerol stock and transferring the cells to the media by swirling the inoculating loop.
4. Secure the lids of the vessel and incubate overnight with shaking at appropriate temp (usually 37°C)

Plasmid Purification:

1. If a glycerol stock of the bacteria has not previously been made do so now by cutting the end off of a 1000μL tip and pipette 200μL of 100% autoclaved glycerol into a cryomicrofuge tube.
   • Glycerol is very viscous and this will take practice to transfer 200μL accurately.
   • Do not pipet from the common glycerol bottle! Always pour some glycerol into a microfuge tube and keep your stock separate
2. Pipette 800μL of overnight culture into the cryovial and mix by swirling the tip and carefully pipetting up and down. Be careful not to suck any media into the barrel of you pipette.
3. Freeze the cells in liquid nitrogen and store in the -800°C freezer
4. Transfer the remaining bacteria into a falcon tube and centrifuge at 5000XG for 7 minutes. Alternately you can pipette cells 1.5 mL at a time in a microfuge tube at max speed for 1.5 minutes.
5. Remove the supernatant and dispose of in the bacteria waste vessel and proceed to the miniprep protocol found in the miniprep kits.

Restriction Digestion

1. Pipet in this order into a 1.5ml microcentrifuge tube:
   1. MilliQ H₂O - final total volume: 20μL
   2. 10x Buffer (corresponding to enzyme) - 2μL
   3. Plasmid DNA (volume according to concentration) 2μg(end concentration < 0.3μg/μL)
   4. Restriction enzyme - 1-2U/μg DNA
      • (Volume according to concentration stated on enzyme tube)
      • (Don’t pipet less than 0.25μL)
   5. Attention with handling restriction enzymes: take them only immediately before use out of -20°C fridge, store on ice, put back into fridge as soon as possible.
2. Incubate at optimal for 1 hour (some enzymes need longer times E.g. SalI) (incubation at 37°C in water bath, but e.g. for SmaI 25-30°C).
3. Take sample for agarose gel or store for short-term on ice or for long-term at -20°C.

Preparative:

1. Parts:
   • MilliQ - 30μL
   • 10x Buffer (corresponding to enzyme) - 1/10 of final volume 3μL
   • Plasmid DNA - 0.2μg/μL, 6μg
   • Restriction enzyme - 1U/μg DNA
2. Incubate 2 hours at 37°C
3. Note:
   • SmaI needs to be incubated at 25-30°C (only 50% activity at 37°C)
   • SalI needs long incubation times (>4h)

Miniprep Using BioBasic EZ-10 Spin Column Kits (High Copy Number Plasmid)

Procedure:

1. Warm Elution Buffer in incubator or in water bath at 37-50°C.
2. Add 1.5mL of the overnight culture to a 1.5mL microcentrifuge tube and centrifuge at 12000rpm for 2 minutes. Drain supernatant and repeat until all of the overnight culture is used.
3. Add 100μL of Solution 1 to the cell pellet, mix by pipetting, let stand 1 minute.
4. Add 200μL of Solution 2 to the mixture, mix gently by inverting the tube 4-6 times and let stand for 1 minute. Do not vortex.
5. Add 350μL of Solution 3 and mix gently by inverting the tube 4-6 times and let stand for 1 minute.
6. Centrifuge at 12000rpm for 5 minutes.
7. Transfer supernatant to an EZ-10 Spin Column and centrifuge at 10000 rpm for 2 minutes.
8. Discard the flow through in the tube. Add 500μL of Wash Solution to the column and centrifuge at 10000 rpm for 2 minutes.
9. Repeat wash procedure in Step 7.
10. Discard the flow though in the collection tube. Centrifuge at 10000rpm for an additional minute to remove any residual wash solution.
11. Transfer the column to a clean, labelled 1.5 mL microcentrifuge tube. Add 50μL of Elution Buffer to the centre part of the column and incubate at room temperature for 2 minutes. Centrifuge at 10000 rpm for 2 minutes.
12. Store purified DNA at -20°C.

Labeling:

All tubes are to be labeled with this information:

• Name
• Date
• miniprep
• Part Number and Plasmid

Transformation of Competent Cells

1. Thaw 20 μL of pre aliquotted cells (Dh5α or BL21 DE3) on ice. Competent cells are stored at -80°C. (Often cells are frozen as 50 μL aliquots – split under sterile conditions for 2 transformations.)
2. Gently pipet maximum 2.0μL (better 1.8μL) of DNA into the competent cells and pipet once up and down to rinse the tip.
   • Never use more DNA than 10% of the volume of the competent cells, otherwise the cells get destroyed by osmotic shock.
3. Mix the DNA into the cells by swirling the tip in the solution.
4. Incubate the cells on ice for 30 minutes.
5. Heat shock the cells in a water bath at 42°C for exactly 45 seconds.
6. Incubate the cells on ice 1 minute.
7. Add 250 μL of sterile media to the cells and incubate at 37°C for 1 hour with shaking (tape microcentrifuge tube in shaking incubator).
8. Label the LB plates on the outside perimeter:
   1. Your name
   2. Date
   3. Organism
   4. Cell strain (e.g. DH5α, BL21DE3 etc.)
   5. Plasmid
   6. Volume plated
9. Plate 100 μL and 50 μL on pre-warmed LB plates containing the appropriate antibiotic. For ligations: plate all 250 μL on (1 or) 2 plate.
10. Leave plate for 10-15 minutes to soak the cell suspension into the agar.
11. Flip plate over (agar on top).
12. Incubate the plates in the 37°C oven overnight.
13. Keep the remaining solution in the 4°C fridge overnight until transformation has been confirmed.

Osmotic Shock Procedure:

Osmotic Shock Procedure:

Note: Osmotic shock procedure should be performed on cells immediately after harvesting (i.e. do not freeze cells).

1. Wash harvested cells with 40 mL ice-cold Wash Buffer (30 mM NaCl, 10 mM Tris-HCl (pH 7.5 @ 4°C)) per gram of harvested cells. After washing, pellet cells by centrifugation (5000 × g, 4°C, 10 min) and remove supernatant.
2. Repeat step 1) once.
3. Resuspend cell pellet in 40 mL Buffer A (33 mM Tris HCl (pH 7.5 @ 20°C)) per gram of harvested cells (perform at room temperature).
4. Rapidly mix suspension with 40 mL Stage I Buffer (40% sucrose, 33 mM Tris-HCl (pH 7.5 @ 20°C), 100 μM ethylenediaminetetraacetic acid (EDTA)) per gram of harvested cells (perform at room temperature).
5. Incubate mixture on a rotary shaker (180 RPM) at room temperature for 10 minutes.
6. Centrifuge mixture (13 000 × g, 4°C, 10 min) and remove supernatant.
7. Rapidly resuspend cell pellet in 80 mL ice-cold Stage II Buffer (500 μM MgCl₂) per gram of harvested cells.
8. Incubate mixture on a rotary shaker (180 RPM) at 4°C for 10 minutes.
9. Centrifuge samples (13 000 × g, 4&degC, 10 min) and remove supernatant containing periplasmic proteins.

Characterization:

• Characterize samples collected throughout the purification by SDS-PAGE. There are also protein markers present in E. coli that can be used to determine the success of the Osmotic Shock Procedure. These markers are β-galactosidase (Cytosolic: 116 kDa) and β-lactamase (Periplasmic: 29 kDa).

References:

• Gray, G. L., Baldridge, J. S., McKeown, K. S., Heyneker, H. L., & Chang, C. N. (1985). Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable. Gene, 39(2), 247-254
• Nossal, N. G., & Heppel, L. A. (1966). The release of enzymes by osmotic shock from Escherichia coli in exponential phase. Journal of Biological Chemistry, 241(13), 3055-3062
• Willsky, G. R., & Malamy, M. H. (1976). Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli. Journal of Bacteriology, 127(1), 595-609

Silver Stain

• Detection limit 0.1-1 ng/band
• Work carefully, you are handling toxic solutions!
• *For two gels. Halve for one gel.*

Solutions:

Use MilliQ H₂O for everything otherwise your gel might turn out brown.

Slot Blot

If not stated otherwise, all steps are performed at room temperature.

1. Incubate 3 Whatman paper for a few minutes in TBS buffer with gentle shaking.
2. Incubate nitrocellulose membrane for 5 minutes in TBS buffer with gentle shaking. (Be sure to mark the top of the membrane with a pencil)
3. Assemble blot:
   • Nitrocellulose membrane
   • 3 Whatman paper
4. Assemble slot blot apparatus.
5. Wash all slots with 100 μL of TBS buffer.
6. Load Samples (usually 100 μL).
7. Wash all slots with 200 μL.
8. Disassemble apparatus and allow membrane to dry for a few minutes.
9. Incubate membrane with blocking buffer (3% (w/v) powdered milk in TBS) for 1 hour.
10. Wash 2 times in TBS (~ 20 - 50 mL)
11. Incubate with 30 ml primary antibody (1:10 000 dilution in 3% milk, TBS) overnight. (See below for handling antibody)
12. Carefully decant antibody back into clean falcon tube and store for at -20°C future use!
13. Wash 2 times for 10 min in TBS-Tween/Triton
14. Incubate with 50 ml secondary antibody (1:5000 in 3% milk, TBS) for 1hour or longer
15. Wash 1 time with TBS-Tween/Triton

Handling of Primary Antibodies:

• Primary Antibodies are expensive; always handle with great care!
• Dilute primary antibody in Blocking Buffer. Dilute Primary antibody (mouse anti-His) in 3% milk(w/v), TBS 1:50000. Dilute Secondary antibody (rabbit anti-mouse) in 3% milk (w/v), TBS 1:5000
• Antibodies can be used multiple times! Be very careful not to spill antibody. After use, place back into original falcon tube and store at -20°C.
• Never use a new antibody unless your supervisor told you to do so.

Detection using Luminol Reaction (in Dark Room):

1. Add 220μl p-Cumaric acid solution to 50ml Luminol solution.
2. Mix Luminol solution and 10 ml H₂O₂ solution.
3. Incubate blots for 5min in the mixture.
4. Lay blot face down on the Typhoon with a transparency.
5. Make the following selections on the computer:
   • Chemiluminescence
   • Press sample
   • For high resolution – 200 microns
   • Select on the grid where the membrane resides and its orientation

Buffers:

TBS

• 10 mM Tris-HCl, pH 7.5
• 150 mM NaCl

TBS-Tween/Triton

• 10 mM Tris-HCl, pH 7.5
• 150 mM NaCl
• 0.05 % (v/v) Tween 20
• 0.2 % (v/v) Triton X-100

For Luminol Reaction:

Luminol Solution

• 22.5 mg Luminol (3-Aminophthalhydrazid) dissolved in 500 μL DMSO
• Diluted in 50 mL 100 mM Tris-HCl, pH 8.5

p-Cumaric Acid Solution

• 15 mg p-cumaric acid in 1 mL DMSO

H₂O₂ Solution

• 20 μL 30% H₂O₂ in 10mL 100 mM Tris-HCl, pH 8.5

100 mM Tris-HCl, pH 8.5

Purification of Polyhistidine Tagged Protein

Buffers and Solutions

Method:

1. Assemble chromatography column (falcon tube)
2. Degas all the buffers
3. Mix bottle of resin and carefully poor the appropriate volume of Ni2+ resin slurry into the column (remember that the slurry is a mixture of solid phase and EtOh) (Ni2+ affinity resins will typically bind 8-12mg of Protein per ml of resin). Avoid frothing: it will result in air bubbles being trapped in the resin.
4. Spin the column at 500g for 2min. Remove the supernatant.
5. Wash the column with 3 column volumes of sterile water. (Gently mix the solid phase with the liquid to create slurry. Spin at 500g for 2min)
6. Wash the column with 6 column volumes of binding buffer and divide into 4 falcon tubes. The column is now ready for binding.
7. Remove 50 μL of cell lysate and store in microfuge tube. Apply the cleared cell lysate (S30) to the 4 columns evenly and gently mix thoroughly. Incubate on ice for 30-60 min inverting periodically to bind protein to the column. ALL FURTHER STEPS ARE PERFORMED AT 4°C
8. Transfer all the column material into 1 falcon tube by spinning 1 column at 500g for 2min removing the supernatant and filling with cleared lysate+resin from another tube, repeat. Remove and store supernatant at 4°C. Take 50uL sample and store at 4°C.
9. Wash the column 2 times with a full falcon tube volume of Buffer A. Pool the washes, store at 4°C and take a 50μL sample.
10. Wash the column 3 times with a full falcon tube volume of Buffer B. Pool the washes, store at 4°C and take a 50μL sample. This step removes the unspecific proteins bound to the column
11. Elute the protein overnight with shaking
12. Run an SDS-PAGE on the samples to ensure proper protein purification.
13. The column material can be reused and is regenerated by washing with 0.2M acetic acid in 30% glycerol. It can be stored as 50% slurry in 30% ethanol to prevent microbial growth.

• Column volume = ~5 ml
• Falcon volume = ~45-50 ml

Tris-tricine Gel:

These gels use tricine instead of glycine that is used in SDS-PAGE. Tricine improves separation and resolution of small proteins. Note, that instead of using one running buffer, there are two different buffers for cathode and anode. All other steps are the same as in the general SDS-PAGE protocol.

Buffers:

3X Gel Buffer:

• 3M Tris-HCl (pH 8.45)
• 0.3% SDS

10X Cathode Buffer:

• 1M Tris (pH 8.25) *No need to correct the pH
• 1M tricine
• 1% SDS

10X Anode Buffer:

• 2M Tris-HCl (pH 8.9)

(You need ~650 mL 1X anode buffer and ~180 mL 1X cathode buffer for each run)

Gel Composition:

Note: Since our protein ladders don’t contain small molecular weight proteins, it is very useful to find a protein sample of a small protein to run as comparison on the gel.

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