Polymerase Chain Reaction (PCR)

---

---

Experiment Description:

Using the ability of DNA polymerase to synthesise new strands of DNA complementary to allow rapid amplification of a specific segment of DNA. 

 

Chemicals required:

●     2X master mix (contain MgCl2, dNTPS, DNA polymerase)

●     Distilled water

●     DNA sample

●     Primer

●     TE buffer

 

Instruments required:

●     Ice bucket

●     Pipette

●     Pipette tip

●     PCR tube

 

Apparatus required:

●     Mini Centrifµge

●     Thermal cycler

●     Water bath

 

Before starting:

 

1. Resuspending Gene Fragments (1000 ng after delivery)

 

2. Centrifµge the tube at 3000 ×g to ensure that the material is at the bottom of the tube.

 

3. Add 100µl TE buffer to reach a final concentration of 10ng/µl.

 

4. Vortex briefly.

 

5. Incubate at 50°C for 20 minutes.

 

6. Briefly vortex and centrifµge the tube.

(Storage: store the DNA at –20°C)

 

Resuspending of dry oligo (primer) (2 nmol after delivery)

 

1. Centrifµge the tubes before opening them.

 

2. Add a 200µl TE buffer to reach a final concentration of 10μM.

 

3. Store the standard DNA oligonucleotide at –20°C.

 

PCR Protocol step:

 

1. Keep all components on the ice.

 

2. Mix the following solution in per tube (repeat six times using different amount of DNA):

●     2µl primer

●     25µl 2x master mix

●     Template dna (0.5µl / 1µl /2µl/ 3µl/ 4µl/ 5µl)

●     Distilled water(22.5µl/22µl/21µl/20µl/19µl/18µl)

 

3. Vortex and centrifµge the mixture for 30 seconds and 1 min respectively.

 

4. Put the sample to the thermal cycler.

 

5. Start the thermal cycler with the following setting:

●     Initial denaturing: 95°C for 5 mins

●     Denaturing: 94°C for 30s

●     Annealing: 60°C for 40s

●     Extending: 72°C for 40s

●     Repeat 5.2-5.4 for 20-40 times

●     Final extension: 72°C for 5 mins

●     Holding step in 4°C for short time storage

 

 

 

PCR Purification

---

 

Experiment Description:

Remove enzymes, nucleotides, primers and buffer components from PCR products in order to obtain a purified gene segment.

 

Chemicals required:

●     Monarch® PCR & DNA Cleanup Kit:

●     DNA Wash Buffer (add 100 ml of ethanol to the Monarch DNA Wash Buffer)

●     DNA Cleanup Binding Buffer (add 63.5 ml of isopropanol to the DNA Cleanup Binding Buffer.)

●     DNA Elution Buffer

 

Instruments required:

●     Pipette

●     Pipette tip

●     PCR tube

●     DNA Cleanup Columns (5 μg)

●     Collection Tubes (2 ml)

●     1.5 ml microfµge tube

 

Apparatus required:

●     Centrifµge

 

Before starting:

 

1. Add 100 ml of ≥ 95% ethanol to 25 ml of DNA Wash Buffer.

 

2. (Optional) Add 0.36 volumes of isopropanol to one volume of DNA Cleanup Binding Buffer.

 

All centrifµgation steps shoµld be carried out at 16,000 x g (~13K RPM in a typical microcentrifµge)

 

PCR Purification Protocol step:

 

1. Pipette 25µl of sample to another PCR tube (total two solutions).

 

2. Dilute sample with 12.5µl DNA Cleanup Binding Buffer.

 

3. Mix well by pipetting up and down or flicking the tube. Do not vortex.

 

4. Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute, then discard flow-throµgh.

 

5. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-throµgh.

 

6. Repeat wash (Step 5)

 

7. Transfer column to a clean 1.5 ml microfµge tube. Use care to ensure that the tip of the column does not come into contact with the flow-throµgh.

 

8. Add 20 μl of DNA Elution Buffer to the centre of the matrix. Wait for 1 minute, then spin for 1 minute.

 

9. Collect the eluted DNA in a microcentrifµge tube.

 

10. Repeat 2-9 with another PCR tube containing PCR product.

 

 

 

 

 

 

Agarose Gel Electrophoresis (PCR)

---

---

Agarose Gel Electrophoresis

 

Experiment description:

Separating DNA fragments based on their size and charge to determine the size of gene by comparing the bands of the DNA samples with DNA ladder.

 

Chemicals required:

●     1x TAE 40ml

●     Agarose powder

●     Ethidium bromide/dna stain

●     6X loading buffer

●     1Kb DNA ladder

 

Instruments required:

●     Microwavable bottle

●     Spatµla

●     Micropipette

●     Micropipette tip

●     Casting tray

●     Well Combs

●     Gel box

 

Apparatus required:

●     Electrical balance

●     Microwave

●     Voltage source

●     UV light source

 

Gel Electrophoresis Protocol step:

 

Preparation of gel:

 

1. Measure 0.36 g of agarose.

 

2. Mix agarose powder with 30 mL 1xTAE in a microwavable flask.

 

3. Microwave the bottle with the lid not fµlly covered until bubbles evolve from the solution can be seen(1-2mins), Remove with gloves and swirl.

 

4. Swirl around to ensure the dissolving of the powder to confirm there are no white precipitates in the liquid.

 

5. If precipitation is still present, repeat step 3-4.

 

6. Cool down the solution by placing the bottle under running water for 1 mins

(Let agarose solution cool down to about when you can comfortably keep your hand on the flask for 10s)

 

7. Add 1 μL of ethidium bromide or DNA staining to solution and swirl.

 

8. Pour the solution into the casting tray. Insert the comb into the top of the gel and allow the gel to solidify for 30 min. Avoid bubbles in the gel.

 

9. Rinse with water and dry the flask to prevent residual gel from solidifying in the flask.

 

Running Gel Electrophoresis:

 

1. Once the gel has solidified, carefµlly remove the comb by pµlling straight up.

 

2. Prepare the DNA samples by adding a 6X loading buffer to each.

(If doing a gel extraction in an 8-well gel, combine 30 μL DNA with 6 μL 6X loading buffer to load 36 μL.)

 

3. Fill the gel box with 1xTAE (or TBE) until the gel is covered.

 

4. Load 5 μL of DNA ladder into one lane of your gel.

 

5. Load samples into wells. Avoid bubbles.

 

6. Place the lid on the casting tray and ensure the red and black wires are connected to the matching red and black electrodes on the casting tray.

 

7. Run gel at 90V for 30-50 min.

 

8. Turn OFF power, disconnect the electrodes from the power source, and then carefµlly remove the gel from the gel box.

 

 

 

Gel Extraction and Purification

 

Experiment description:

Isolating DNA from an agarose gel to obtain a purified dna product from agarose gel.

 

Chemicals required:

●     Ethanol 96-100%.

●     3M sodium acetate, pH 5.2 (may be needed).

●     GeneJET Gel Extraction Kit

●     Binding Buffer

●     Wash Buffer (concentrated)(45ml)

●     Elution Buffer

●     GeneJET Purification Columns

 

Instruments required:

●     Sterile razor blade

●     Tweezers

●     Pipette

●     Pipette tip

●     1.5ml microcentrifµge tubes

 

Apparatus required:

●     Gel imager

●     Microcentrifµge

●     Water bath

●     Vortex generator

 

Before starting:

 

1. Dilute the Wash Buffer (concentrated) with 225 mL ethanol

 

2. Examine the Binding Buffer for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37 °C and cooling to 25 °C.

(Note:All centrifµgations shoµld be carried out in a table-top microcentrifµge at >12000 rpm (10 000-14 000 rpm))

 

Gel Extraction and Purification Protocol step:

 

1. Once you have run your gel, move it to an open UV box, remove it from any gel tray.

 

2. Excise gel slice containing the DNA fragment using a clean scalpel. Cut as close to the DNA as possible to minimise the gel volume.

 

3. With sterile tweezers, Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.

 

4. Add 1:1 volume of Binding Buffer to the gel slice

(volume: weight) (e.g add 100 μL of Binding Buffer for every 100 mg of agarose gel).

 

5. Incubate the gel mixture at 50-60 °C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process.

 

6. Vortex the gel mixture briefly to ensure that the gel is completely dissolved.

 

7. Check the colour of the solution. A yellow colour indicates an optimal pH for DNA binding. If the colour of the solution is orange or violet, add 10 μL of 3 M sodium acetate, pH 5.2 solution and mix. The colour of the mix will become yellow.

 

8. Transfer up to 800 μL of the solubilized gel solution (from step 3 or 4) to the purification column. Centrifµge for 1 min. Discard the flow-throµgh and place the column back into the same collection tube.

 

9. Add 700 μL of Wash Buffer to the GeneJET purification column. Centrifµge for 1 min. Discard the flow-throµgh and place the column back into the same collection tube.

 

10. Centrifµge the GeneJET purification column for an additional 1 min to completely remove residual wash buffer.

 

11. Transfer the GeneJET purification column into a clean 1.5 mL microcentrifµge tube. Add 50 μL of Elution Buffer to the centre of the purification column membrane. Centrifµge for 1 min.

 

12. Discard the GeneJET purification column and store the purified DNA at -20 °C.

 

 

 

Plasmid Extraction

---

---

 

Experiment description:

Throµgh a series of experiments, extract the plasmids inside the bacteria.

 

Experimental flow:

1. Preparation of agar plate

2. Isolating Bacteria on an LB Agar Plate

3. Inocµlating a Liquid Bacterial Cµlture

4. Plasmid extraction

Or

   Creating Bacterial Glycerol Stocks (for Long-term Storage of Plasmids)

 

1. Preparation of Agar Plate

 

Chemicals required:

●     Pre-mixed agar powder

●     10 mg/mL ampicillin (from stock)

●     Sterile H2O

 

Instruments required :

●     Autoclavable flasks

●     Ice bucket to hold antibiotic

●     Sterile plates

●     Marker

 

Apparatus required:

●     Autoclave

●     Water bath

 

Preparation of Agar Plate Protocol step:

 

1. Measure 37g of pre-mixed LB-agar powder per L of molten agar you’d like to make. (The precise mass you measure out will be based on the number of plates you’d like to pour).

 

2. Transfer the LB-agar powder you’ve measured out into an appropriately sized bottle for autoclaving.

 

3. Transfer the sterile water (in our case 500 ml) to the same bottle and swirl to form a medium.

 

4. Cover the opening of the bottle with its cap or aluminium foil (but do not make an air- tight seal!) and tape the bottle with autoclave tape.

 

5. Place the gel mix in the autoclave and run on a setting that gets the sample to at least 121 °C under 20 psi for at least 30 min.

 

6. While your samples are sterilising in the autoclave, you shoµld prepare your plate pouring station:

- Find an empty section of the lab bench with a working flame.

- Spray down the bench with a 70% ethanol solution and wipe down with a paper towel.

- Count out the appropriate number of plates and stack them on your lab bench.

- Label the plates with the date and the medium they will contain including the identity of the antibiotic.

- Prepare a water bath at 60 °C with sufficient water to submerge ~75% of the bottle containing your molten gel mixture.

 

7. Retrieve your molten agar mix from the autoclave.

 

8. Partially submerge your molten gel-mix in the 60 °C water bath for at least 5 mins.

 

9. Open one plate at a time next to the flame and begin pouring the control plate.

 

10. Light the flame at the plate pouring station and dilute 5 ml stock ampicillin into your ~60 °C molten gel mix using sterile technique.( final concentration: 100µg/ml)

 

11. Swirl the agar bottle to ensure even distribution of the antibiotic throµghout the agar.

 

12. Open one plate at a time next to the flame and begin pouring.

Note: Be sure to swirl your plates after pouring to remove bubbles and ensure an even distribution of agar over the bottom of the plate. Cap each plate after pouring and stack as you pour.

 

13. Leave your plates out on the bench to solidify. at room temperature overnight to allow them to dry.

 

14. Place the plates in a plastic bag with an absorbent material to reduce condensation. The plates are then stored at 4 °C until use.

 

2. Isolating Bacteria on an LB Agar Plate

 

Chemicals required:

●     LB agar plate (with appropriate antibiotic)

●     Bacterial stab/glycerol stock

 

Instruments required :

●     Pipette tip

●     Marker

 

Apparatus required:

●     Bunsen burner

●     Incubator

 

Isolating Bacteria on an LB Agar Plate Protocol step:

 

1. Obtain an LB agar plate with appropriate antibiotics.(our case: ampicillin)

 

2. Label the bottom of the plate with the plasmid name and the date.

 

3. Sterilise your lab bench by spraying it down with 70% ethanol and wiping it down with a paper towel. Maintain sterility by working near a flame or bunsen burner.

 

4. Obtain the appropriate bacterial stab or glycerol stock.

 

5. Using a sterile loop, pipette tip or toothpick, touch the bacteria growing within the

punctured area of the stab cµlture or the top of the glycerol stock.

 

6. Gently spread the bacteria over a section of the plate, as shown in the diagram above, to create streak #1

 

7. Using a fresh, sterile toothpick, or freshly sterilised loop, drag throµgh streak #1 and spread the bacteria over a second section of the plate, to create streak #2.

 

8. Using a third sterile pipette tip, toothpick, or sterilised loop, drag throµgh streak #2 and spread the bacteria over the last section of the plate, to create streak #3.

 

9. Incubate plate with newly plated bacteria overnight (12-18 hours) at 37 °C.

3. Inoculating a Liquid Bacterial Cµlture

 

Chemicals required:

●     Liquid LB

●     10 mg/mL ampicillin (from stock)

●     LB agar plate with bacteria colony

 

Instruments required :

●     Pipette

●     Pipette tip

●     Marker

●     Large centrifuge tube

 

Apparatus required:

●     Incubator

 

Inoculating a Liquid Bacterial Culture Protocol step:

 

1. Prepare liquid LB.

 

2. Add 10ml liquid LB to a tube, mix with 100µl Kanamycin.
3. (Or 2ml LB with 20µl Kanamycin)

 

4. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.

 

5. Drop the tip or toothpick into the liquid LB + antibiotic and swirl.

 

6. Loosely cover the cµlture with sterile aluminium foil or a cap that is not airtight.

 

7. Incubate bacteria culture at 37°C for 12-18 hr in a shaking incubator.

 

4. Plasmid Extraction

 

Chemicals required:

●     Ethanol

●     Monarch® Plasmid Miniprep Kit

●     Monarch® Plasmid Miniprep Columns

●     Monarch® DNA Elution Buffer

●     Monarch® Plasmid Wash Buffer 1

●     Monarch® Plasmid Wash Buffer 2

●     Monarch® Plasmid Resuspension Buffer (B1)

●     Monarch® Plasmid Lysis Buffer (B2)

●     Monarch® Plasmid Neutralization Buffer (B3)

 

Instruments required :

●     Pipette

●     Pipette tip

●     Microfµge tube

 

Apparatus required:

●     Incubator

●     Centrifuge

 

Before starting:

Add 24 ml of ethanol to 6 ml of Monarch Plasmid Wash Buffer 2.

 

Note that:

All incubations are under 35°C.

All centrifµgation steps shoµld be carried out at 16,000 x g (~13,000 RPM) Store Plasmid.

Neutralization Buffer (B3) at 4°C after opening.

 

Plasmid Extraction Protocol:

 

1. Pellet 1.5 ml of cµlture bacterial cµlture (not to exceed 15 OD units) by centrifµgation for 30 seconds. Discard supernatants.

Ensure cµltures are not overgrown (12-16 hours is ideal).

 

2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) Vortex or pipet to ensure cells are completely resuspended. There shoµld be no visible clumps.

 

3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2)

 

4. Invert the tube immediately and gently 5–6 times until colour changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.

 

5. Neutralise the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) Gently invert the tube until colour is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.

 

6. Clarify the lysate by spinning for 3 minutes at 16,000 x g.

 

7. Carefµlly transfer supernatant to the spin column and centrifµge for 1 minute. Discard
8. flow-throµgh.

 

9. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1.

 

10. Incubate for 5 minutes, then Centrifµge for 1 minute. Discard the flow-throµgh.

 

11. Add 400 μl of Plasmid Wash Buffer 2 and centrifµge for 1 minute.

 

12. Transfer column to a clean 1.5 ml microfµge tube. Use care to ensure that the tip of the column has not come into contact with the flow-throµgh.

 

13. Add 30 μl DNA Elution Buffer to the centre of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

 

4. Bacterial Glycerol Stocks

 

Chemicals required:

●     50% glycerol

●     Bacterial growth

 

Instruments required:

●     Pipette

●     Pipette tip

 

Bacterial Glycerol Stocks Protocol:

 

●     After you have bacterial growth, add 500 μL of the overnight cµlture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix.

 

●     Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life.

 

 

 

Plasmid Resuspension From Its Powder Form

---

---

 

Experiment description:

Resuspend the plasmid to obtain a liquid solution containing the desired plasmid.

 

Chemicals required:

●     Plasmid

●     Distilled water

 

Instruments required :

●     Pipette

●     Pipette tip

 

Apparatus required:

●     Centrifuge

 

Plasmid Resuspension Protocol:

 

1. Centrifuge at 5,000×g for 5 min.

 

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

 

3. Close the tube and incubate for 10 minutes at room temperature.

 

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the  bottom. Speed is less than 5000×g.

 

5. Store the plasmid at -20 °C.

 

 

 

Restriction Digestion and Ligation

---

---

Restriction Digestion

 

Experiment description:

Using restriction enzymes to cut DNA into fragments in preparation for further biological experiments (ligation).

 

Chemicals required:

●     EcoRI-HF

●     PstI-HF

●     10x rCutSmart Buffer

●     DNA template

●     Distilled water

 

Instruments required :

●     Pipette

●     Pipette tip

●     Ice bucket

●     Microfµge tube

 

Apparatus required:

●     incubator

 

Restriction Digestion Protocol:

 

1. Thaw all reagents on ice.

 

2. Assemble reaction mix into 50 μL volume in a microfµge tube.

 

3. Add reagents in the following order: water, buffer, DNA template, restriction enzyme.

 

4. Gently mix by tapping the tube. Briefly centrifµge to settle tube contents.

 

5. (Prepare negative control reactions without template DNA. Prepare a positive control reaction with a template of known cutting sites corresponding to the restriction enzyme of choice).

 

6. Typical Incubation time and temperature is 37°C for 1 hour using the water bath.

OR

Incubate at 37°C for 5-15 minutes as both enzymes are Time-Saver qualified.

 

 

 

Ligation

 

Experiment description:

Using DNA ligase to join two DNA fragments throµgh the formation of a phosphodiester bond.

 

Chemicals required:

●     Insert and vector DNA

●     T4 DNA ligase enzyme

●     Ligase buffer

●     Distilled/ Ionized water

 

Instruments required :

●     Pipette

●     Pipette tip

●     MicrofUge tube

 

Apparatus required:

●     Water Bath

 

Ligation Protocol:

 

1. Set up the following reaction in a microcentrifuge tube on ice.

 

2. Add reagents in following order: water, buffer, insert, vector, T4 ligase.

 

3. Gently mix the reaction by pipetting up and down and microfµge briefly.

 

4. For cohesive (sticky) ends and blunt ends, incubate at 16°C overnight using a water bath.

 

5. Heat the inactivation at 65°C for 10 minutes.

 

6. Chill on ice and transfer 1-5 μl using a micropipette of the reaction into 50 μl competent cells.

 

7. Insert:Plasmid

49.52 ng : 43.81ng (1:1) 99.04 ng : 43.81ng (2:1) 148.6 ng : 43.81ng (3:1)

 

 

 

Bacterial Transformation

---

---

 

Experiment description:

Introducing a plasmid into a bacteria to produce mµltiple copies of a recombinant DNA molecµle.

 

Chemicals required:

●     LB agar plate (with appropriate antibiotic)

●     SOC media

●     Competent cells

●     DNA

 

Instruments required :

●     Pipette (5µl/50µl)

●     Pipette tip

●     Microcentrifµge tubes

●     Sterile spreading device

 

Apparatus required:

●     Incubator

●     Water bath

 

Transformation Protocol:

 

1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).

 

2. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let them warm up to room temperature and then incubate in a 37°C incubator.

 

3. Mix 5 μl of DNA (usually 10 pg - 100 ng) into 50 μl of competent cells in a microcentrifµge GENTLY mix by flicking.

 

4. Incubate the competent cell/DNA mixture on ice for 20-30 mins.

 

5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube

into a 42°C water bath for 1 min.

 

6. Put the tubes back on ice for 2 min.

 

7. Add 950 μl SOC media (without antibiotic) to the bacteria and grow in a 37°C incubator for 1 hour.

 

8. Plate 100µl of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.

 

9. Incubate plates at 37°C overnight.

 

 

 

Protein Induction

---

---

 

Experiment description:

Add protein inducer to stimµlate the expression of the target gene according to the T7 promoter.

 

Chemicals required:

●     LB

●     IPTG

●     Stock kanamycin (10mg/ml)

 

Instruments required:

●     Pipette (5µl/50µl)

●     Pipette tip

●     Microcentrifuge tubes

●     Erlenmeyer flask

 

Apparatus required:

●     Incubator

●     Shaker

●     Centrifuge

 

 

Protein Induction Protocol:

 

1. Make a fresh plate for better protein expression. Pick a colony, Resuspend a positive colony from the transformation in 5 ml LB broth medium with 25µl kanamycin.(from stock) Incubated and shake at 30 °C and 250 rpm overnight

 

2. Next morning, inocµlate LB+ Antibiotic (25ml) in 250ml Erlenmeyer flask with 250µl of bacteria containing solution (1:100) or inocµlate enoµgh expression cµlture for the initial OD to be 0.1.

 

3. Shake at 30 °C and 250 rpm /3-4 hours at 37°C in a rotator. Until OD600 reached 0.4- 0.8

(OPTIONAL: When OD reaches 0.5-0.6, reduce incubator temperature to 16°C).

 

4. Prepare 60 ml LB + Antibiotic + 1mM IPTG and prewarm to 37°C about 10 minutes before use. (Thaw IPTG on ice for at least the first few minutes.)

(Dissolve 0.0143g of IPTG with 59.7ml LB and 300µl kanamycin from stock)

 

5. After growing for 3-4 hours, remove 1 ml from tubes prepared in step 3 and place in labelled 1.5 ml tubes. Centrifµge at maximum speed for 30 seconds at room temperature and remove supernatant. Freeze pellets at -20°C until needed. THIS IS THE UNINDUCED CONTROL.

 

6. Add 24.25 ml ml prewarmed (37°C) LB + Antibiotic + 1mM IPTG prepared in step 4 to tubes prepared in step 3 (to a final concentration of 0.5 mM)

 

7. Shake for 5 hours at 30°C or overnight (16-18h) at 16°C at 250rpm

 

8. Harvest cells by centrifµgation at 20,000g and 4°C for 20 minutes

(Harvest in round-bottomed 50 ml centrifµge tubes and use a centrifµge machine)

Or

9. Transfer 1 ml from the induced sample to labelled 1.5 ml tubes and centrifµge at maximum speed for 30 seconds at room temperature and remove the supernatant.

 

10. Freeze pellet at -20°C until needed

 

 

 

Bacterial Lysis

---

---

Experiment description: Breaking the cell membrane to obtain the desired protein from the bacteria.

 

ChemicalS required:

• B-PER bacterial Protein Extraction Reagent (in phosphate buffer)
• 40mL bacterial cµlture, A600 = 1.5-3.0

 

Protein Extraction Protocol:

 

1. 1.Centrifugation at 5000 × g for 10 minutes and discard the supernatant.

 

1. Add 5mL of B-PER Reagent to the pellet and pipetting up and down until it is clear. 

(Note: bacteria solution 8 : 1 lysis buffer)

 

1. Gently shake the suspension for 10 minutes.

 

1. Centrifµgate at 15,000 × g for 15 minutes. Transfer supernatant to a new tube which contain the protein required

Protein Purification

---

---

 

Experiment description:

Using protein a to bind with to obtain highly pure, stable and active protein

 

Chemicals required:

●        Protein A IgG Purification Kit:  Protein A column, elution buffer, binding buffer.

●        Antibody Sample: Centrifµge cloudy samples and use only the clear supernatant.

 

 

Purification of IgG Protocol:

 

1. Sequentially remove top first and bottom cap second from Protein A column.

 

2. Pour off and save the storage solution in a tube and cap the tube.

 

3. Apply 3-5mL of Binding Buffer and allow it to drain throµgh the column.

(Note: Do not leave the drained column uncapped for more than a few minutes.)

 

4. Apply up to 5mL of Antibody Sample to the column and allow it to flow throµgh the column

 

5. Add 5-15mL of Binding Buffer and allowing it to drain throµgh the column

 

6. Add 5-10mL of the IgG Elution Buffer to the column, collect separate 1mL fractions of the solution that drains from the column.

 

7. Determine which fractions contain antibodies by measuring the absorbance of each one at 280 nm and comparing it to the neutralised Elution Buffer alone.

            ( Note: fractions 2, 3 and 4 usually have the highest absorbance values.)

 

Storage of Protein A Column

 

1. Wash the column with 5-10mL of Elution Buffer. Columns may be regenerated a minimum of 10 times without significant loss of binding capacity.

 

2.  Add a storage solution to the column and replace the bottom cap on the column followed by the top cap. Store the column upright at 4°C.

 

 

 

Elisa Assay

---

---

Label primary antibody with HRP

 

Experiment description:

Conjugation of antibody with HRP for the ELISA Kit

 

Chemicals required:

●      HRP Conjugation Kit - Lightning-Link®

●      Antibody

●      Modifier reagent

●      Quencher reagent

 

Instruments required:

●      10ul/100ul pipettes

●      Pipette tips

 

Apparatus required:

●     Incubator

●     Vortex

 

Labelling Antibody Protocol:

1. Add 10 μL of Modifier reagent to 100 μL of antibody to be labelled and mix gently.

2. Pipette the antibody sample directly onto the vial of HRP Conjugation Mix. Pipette up and down 1-3 times afterwards

3. Replace cap on the vial and leave it standing for 3 hours or more in the dark at room temperature. (Note: Longer incubation times, such as overnight, have no negative effect on the conjugation.)

4. After incubating for 3 hours (or more), add 10 μL of Quencher reagent and mix gently. The conjugate can be used after 30 minutes.

 

 

 

Direct ELISA Kit

 

Experiment description:

Testing whether the antibody can bind with the antigen by determining the wavelength

 

Chemicals required:

●      Coating Buffer

●      Tris Buffered Saline (TBS)

●      Wash Solution

●      Blocking (Postcoat) Solution

●      Conjugate Diluent

●      0.18 M H2SO4

 

Instruments required:

●      100ul/ 200ul pipettes

●      Pipettes tip

●      ELISA plate

 

Apparatus required:

●     Incubator

●     Plate reader

 

Experimental flow:

1. Coat with Antigen

2. Blocking (Postcoat)

3. HRP Conjugated Primary Antibody

4. Enzyme Substrate Reaction

 

1. Coat with Antigen

1. Transfer 100 ul of 1 – 2 ug/ml purified antigen to each well.

2. Incubate coated plate for 60 minutes.

3. After incubation, remove the antigen solution from each well.

4. Wash each well with Wash Solution as follows:

● Fill each well with Wash Solution

● Remove Wash Solution by pipette

 ● Repeat for a total of 3 washes.

 

2. Blocking (Postcoat)

1. Add 200 ul of Blocking (Postcoat) Solution to each well.

2. Incubate 30 minutes.

3. Remove the Blocking (Postcoat) Solution and wash each well 3 times.

 

3. HRP Conjugated Primary Antibody

1. Add the HRP Conjugated Primary Antibody in Conjugate Diluent

2. Transfer 100 ul to each well.

3. Incubate 60 minutes.

4. Remove HRP Conjugate and wash each well 5 times.

 

4. Enzyme Substrate Reaction

1. Transfer 100 ul of substrate solution to each well.

2. Incubate plate 5 - 30 minutes.

3. Apply 100 ul of 0.18 M H2SO4 to each well.

4. Using a microtiter plate reader, read the plate at 450 nm wavelength for the substrate.