Digestion of Plasmids and Gene Segments by Restriction Enzymes

During plasmid construction, restriction enzymes are used to cleave plasmid DNA and DNA fragments of the target gene. These enzymes recognize and cut specific DNA sequences, producing complementary sticky or blunt ends. The target gene is then inserted into the plasmid at the cleavage site using DNA ligase to form a recombinant plasmid.

Restriction enzymes: Ecor I and Not I

Plasmid: pPIC9K

Steps:

1.Introduce restriction enzymes cutting sites into both ends of the target fragment using primers.

2.Digest the target fragment and the vector with the same restriction endonuclease.

3.Ligate the fragment of interest with the cleaved vector in vitro.

4.Transform the ligation product into Escherichia coli.

5.Screen for successful recombinants.

DNA Purification

Purify the DNA using a standard DNA purification kit (e.g., DP204).

PCR

Amplify the target DNA using a PCR machine.

Steps:

1.Prepare the PCR reaction mix by combining the following components: DNA template, primers, DNA polymerase, buffer solution, and dNTPs, with an appropriate volume of water.

Primer	1μl
DNA Template	1 μl
Buffer Solution	10 μl
ddH₂O	7 μl

2.Set the temperature program on the PCR instrument according to the required amplification conditions: pre-denaturation at 98°C for 0.5-3 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 15-30 seconds, and final extension at 72°C for 5 minutes.

3.Load the PCR reaction mix into a PCR tube or PCR plate.

4.Start the PCR reaction by placing the PCR tube or plate into the PCR machine and running the temperature program.

5.After the PCR reaction is complete, analyze the target DNA by electrophoresis or other suitable methods.

Agarose Gel Electrophoresis

Steps:

1.Gel Preparation:

• Determine the appropriate agarose concentration based on the DNA size (see table below).

• Weigh the appropriate amount of agarose and dissolve it in 1x TAE buffer.

• Boil in a microwave until the solution is clear.

• Add nucleic acid dye at a ratio of 1:30,000 (e.g., add 1 μl of dye to 30 ml of gel solution).

• Mix well, pour into the gel casting tray, and insert an appropriate comb based on the number and volume of samples.

Gel Concentration (%)	Linear DNA Length (bp)
1.0	500 - 10,000
1.2	400 - 7,000
1.5	200 - 3,000
2.0	50 - 2,000

2.Loading:

• Load the marker (100 bp/1 kbp) first, followed by the amplified target DNA samples in order.

3.Gel Running:

• Connect the electrophoresis apparatus: black to black and red to red.

• Set the voltage to 130 V and run for 40 minutes.

• Start the run by pressing "Run."

4.Observation:

• Visualize the DNA bands using a UV transilluminator.

• Use DNA ladders such as Kang Wei Century 1kb DNA Ladder or Biology 1kb DNA Ladder for size reference.

Purification of Linearized Plasmid

• Use SacI restriction enzyme for linearization.

Reaction Components:

Component	Volume
Plasmid	20 μl (15-20 μg)
Buffer	15 μl
ddH₂O	160 μl
SacI	5 μl

• The total reaction volume is 200 μl.

Notes:

Calculate the plasmid amount using the formula X = m * c / 1000.

Add ddH₂O and Buffer first, followed by the plasmid, and finally the enzyme.

Use an endotoxin-free plasmid extraction kit (e.g., BOLG907).

Chemical Transformation

GS115:

Reagents:

Reagent	Volume	Storage
GS115	-	-80°C
Yeast Transformation Reagent	100 μl	-20°C
Transformation Liquid of Pichia pastoris	5 ml	4°C

Steps:

1.Mix 1.5 μg linearized plasmid, 10 μl yeast transformation reagent, 100 μl natural competence solution, and 500 μl Pichia pastoris transformation liquid in a 1.5 ml EP tube. Mix gently 6-8 times.

2.Incubate at 30°C for 30 minutes, inverting the tube 6-8 times every 10 minutes.

3.Add 20 ml of dimethyl alum at each step.

4.Incubate at 42°C for 15 minutes, inverting the tube 6-8 times every 5 minutes.

5.Centrifuge at 12,000 rpm (immediate stop at 12,000 rpm). Resuspend in 1 ml YPD medium and incubate at 30°C for 1 hour.

6.Centrifuge again at 12,000 rpm. Spread the supernatant onto PBS-filtered plates and incubate at 30°C.

KM71H:

Reagents:

Reagent	Volume	Storage
cDNA	100 μl	-20°C
B2	20 ml	-20°C
B3	11 ml	-20°C

Steps:

1.Add 0.1-5 μg plasmid DNA (5-50 μg linearized plasmid) and 10 μl cDNA to water-melted KM71H cells. Incubate in a 30°C water bath, gently inverting every 15 seconds until cells melt.

2.Add 1.4 ml B2 solution, incubate at 30°C for 6 minutes, inverting every 20 minutes.

3.Centrifuge at 3,000 rpm for 3 minutes, discard the supernatant, and resuspend the pellet in 1 ml B3 solution.

4.Centrifuge again at 3,000 rpm for 3 minutes, discard the supernatant, and resuspend in 100 ml B3 solution.

5.Spread the cells on plates and incubate at 30°C.

PCR

Amplification was performed using a PCR machine.

Steps:

1.Prepare the PCR Reaction Mix:

• Combine the following components in a PCR tube or plate:

a.Primer( Forward/Reverse): 1 μl

b.DNA Template: 1 μl

c.Buffer Solution: 10 μl

d.ddH₂O: 7 μl

e.DNA Polymerase and dNTPs should also be added as per the specific protocol.

• Mix the components thoroughly.

2.Thermocycling conditions:

• Configure the PCR machine according to the amplification conditions:

Step	Temperature	Time	Cycle
Initial Denaturation	98°C	0.5-3 minutes	1
Denaturation	98°C	10 seconds	35
Annealing	60°C	20 seconds
Extension	72°C	15-30 seconds
Final Extension	72°C	5 minutes	1
Hold	4-10°C	Forever	1

3.Load the Reaction Mix:

• Transfer the PCR reaction mix into a PCR tube or plate.

4.Start the PCR Reaction:

• Place the PCR tube or plate into the PCR machine.

• Initiate the temperature program and allow the PCR machine to complete the amplification.

5.Analyze the PCR Product:

• After PCR completion, analyze the amplified DNA using agarose gel electrophoresis or other suitable methods.

Agarose Gel Electrophoresis

Steps:

1.Gel Preparation:

• Determine the agarose gel concentration based on the DNA size (see table below).

• Weigh the appropriate amount of agarose and dissolve it in 1x TAE buffer.

• Heat the solution in a microwave until it becomes clear.

• Add nucleic acid dye at a ratio of 1:30,000 (e.g., add 1 μl of dye to 30 ml of gel solution).

• Mix thoroughly, pour the solution into the gel casting tray, and insert the comb according to the number and volume of samples.

Gel Concentration (%)	Linear DNA Length (bp)
1.0	500 - 10,000
1.2	400 - 7,000
1.5	200 - 3,000
2.0	50 - 2,000

2.Load the Gel:

• First, load the DNA marker (e.g., 100 bp or 1 kbp ladder), followed by the amplified target DNA samples.

3.Run the Gel:

• Connect the electrophoresis apparatus: black electrode to the black terminal and red electrode to the red terminal.

• Set the voltage to 130 V and run for 40 minutes.

• Click "Run" or the corresponding button to start the electrophoresis.

4.Visualize the Gel:

• Observe the DNA bands under a UV transilluminator.

• Use DNA ladders such as Kang Wei Century 1kb DNA Ladder or Biology 1kb DNA Ladder for size reference.

Induced Expression of Product

Steps:

1.Preparation and Activation:

• Mix 600 μl of GS115 solution with 600 μl of 50% glycerol and add 100 μl of this mixture to 5 ml of YPD medium (non-resistant, activated).

• Incubate at 30°C with shaking for 24 hours.

2.Adaptation:

• Add 20 ml of BMGY to the culture and incubate at 30°C with shaking for another 24 hours to adapt the bacteria to the expression environment.

3.Induction:

• Centrifuge the culture at 4,000 rpm for 6-8 minutes. Discard the supernatant and resuspend the pellet in 20-25 ml of BMMY medium.

• Incubate at 30°C with shaking for 24 hours.

4.Induction of Expression:

• Use filter-sterilized 1% methanol for induction. Add methanol every 24 hours.

• Prior to each addition, take 1 ml of the bacterial solution to check methanol solubility and optimize expression conditions.

• Continue the induction process for a total of 96 hours, or until the addition of BMMY is complete.

5.Protein Detection by Western blot:

• Prepare samples for Western blot as follows:

a.Mix 50 μl of β-Mercaptoethanol (freshly prepared) with 5x loading buffer.

b.The final mix should be 100 μl of loading buffer plus 25 μl of β-Mercaptoethanol.

c.Boil the mixture for 10-15 minutes.

d.Allow to cool, then store at -80°C until ready for gel electrophoresis.

Nickel Column Affinity Chromatography:

1.Column Preparation:

• Allow alcohol to drain naturally under gravity.

2.Equilibrate the Column:

• Add three column volumes of Binding Buffer to equilibrate the column.

• Stand for 2-3 minutes, repeating this step several times. Maintain a flow rate of 2 ml/min.

3.Sample Application:

• Apply the sample liquid to the column, controlling the flow rate to approximately 2 ml/min. Pass the sample through the column three times, totaling 15 ml.

4.Washing:

• Wash the column with 6 ml of low concentration imidazole solution. Repeat the washing step three times.

5.Column Cleaning:

• Clean the column with deionized water and store it with alcohol.

Coomassie Brilliant Blue Staining

Preparation of Staining Solution:

• Coomassie Brilliant Blue R250: 2.5 g

• 95% Ethanol: 250 ml

• Glacial Acetic Acid: 80 ml

• ddH₂O: 670 ml

Preparation of Fixing and Decolorizing Solution:

• 100% Ethanol: 500 ml

• Glacial Acetic Acid: 100 ml

• ddH₂O: 400 ml

Gel Preparation

Steps:

Prepare the Gel:

• Ingredients:

a. Acrylamide: 15% for separating gel.

b. Bisacrylamide: For gel polymerization.

c. Sodium Dodecyl Sulfate (SDS):c. Anionic detergent that denatures proteins by disrupting intermolecular hydrogen bonds, thereby unfolding proteins and destroying their secondary and tertiary structures.

d. Tetramethylethylenediamine (TEMED):d.Accelerator for polymerization.

e. Ammonium Persulfate (APS): Catalyst for polymerization.

• Procedure:

a.Prepare the separating gel solution (15%) and ensure no leaks in the gel apparatus by draining the glass plates.

b.Mix with TEMED and APS, then immediately pour the solution into the gel apparatus, carefully avoiding bubbles and debris. Insert the comb 1 cm from the top, ensuring an even distribution of the gel.

c.Allow the gel to polymerize by adding ethanol to the top of the gel (about 200 ml) to prevent polymerization at the top. Leave for 20-30 minutes, then pour out the ethanol and allow the gel to dry.

d.Prepare the stacking gel (2.5-3 ml/block). Anti-foaming agents may be added if necessary. Insert the comb 30 minutes after pouring.

Electrophoresis:

• Electrophoresis Buffer:

a. Composition: a.1000 ml water + 188 g Glycine + 1 g SDS + 30.2 g Tris. The buffer is recyclable.

• Procedure:

a.Fill the electrophoresis tank with the buffer solution and ensure there are no leaks.

b.Remove the comb from the gel and check for uniformity in the wells.

c.Load samples from left to right: add 2.5 μl of protein marker and 8 μl of sample. Fill the space between the gels with electrophoresis buffer.

d.Insert the electrodes (positive and negative) and run electrophoresis at a constant voltage of 80 V with a current of 200 mA. Monitor for bubbles.

e.When the marker bands have migrated appropriately, increase the voltage to 120 V and continue until the blue dye front reaches the bottom of the gel (typically 1-1.5 hours). Maintain the electrophoresis tank in an ice bath if running at higher voltages.

Transfer to Membrane (Western blot):

Steps:

• Prepare Transfer Solution:

a. Solution:800 ml water + 200 ml methanol + Glycine + Tris (quantities as per standard protocols). Keep the solution on ice.

• Prepare the Gel and Membrane:

a.After electrophoresis, carefully transfer the gel into a transfer box. Soak the sponge and filter paper in the transfer solution.

b.Cut a PVDF membrane (polyvinylidene fluoride) without touching the surface. Activate the membrane by soaking in methanol for 20 seconds (ensure the positive side is activated).

• Assemble the Transfer Sandwich:

a.Assemble as follows: Black clip + Sponge + Filter paper + Gel + PVDF membrane + Filter paper + Sponge + Red clip.

b.Place the sandwich into the transfer tank and run at 200 V and 230 mA for 2 hours in an ice bath. For overnight transfers, use 100 mA and ensure proper orientation of electrodes (black to black, red to red).

• Blocking:

Blocking Solution: Mix 1 g skim milk powder with 20 ml 1x TBST.

10x TBST Buffer Preparation: Mix 24.2 g Tris, 88 g NaCl, and 800 ml ddH₂O. Adjust volume to 1 L and store at room temperature.

• Cutting the Membrane:

Select the appropriate antibody and determine the bands of interest.

Cut the membrane based on the target bands and molecular weight. Avoid touching the membrane directly.

Detection of product

• Incubation with Primary Antibody:

Incubate the membrane with the primary antibody (face up) in a shaker at 4°C overnight or at room temperature for 2-4 hours.

After incubation, discard the primary antibody and do not store it. Wash the membrane with TBST (3 times, 5 minutes each) on a shaker.

• Incubation with Secondary Antibody:

Dilute the secondary antibody in TBST.

Incubate the membrane with the secondary antibody for 45 minutes to 1 hour.

Avoid prolonged incubation to reduce background. Rinse the membrane with TBST (3 times, 5 minutes each).

• Development:

In a darkroom, prepare the developer solution by mixing Developer A and Developer B in a 1:1 ratio.

Ensure the membrane is dry but not over-dried.

Develop the membrane face up, avoiding bubbles.