• 10 g/L tryptone
• 10 g/L NaCL
• 5 g/L yeast extract
• 20 g/L agar
• Fill up with water to 1 L
• Autoclave the bottle with medium
• Add 1 ml of 100 mg/mL ampicillin solution into 1 liter medium

• 10 g/L tryptone
• 10 g/L NaCl.
• 5 g/L yeast extract
• Fill up with water to 1 L
• Autoclave the bottle with medium.
• Add 1ml of 150 mg/mL of ampicillin solution into 1 liter medium.

• 12 g tryptone in 900 ml
• 5 g (4 ml) glycerol in 900 ml
• 24 g yeast extract in 900 ml
• Fill up with water to 900 ml
• Autoclave the bottle with medium
• Add 100 ml 10x TB-salt to 900 ml TB media

• 23.1 g/L KH₂PO₄
• 125.4 g/L K₂HPO₄
• Autoclave the bottle.

• 20 g/L tryptone
• 0.584 g/L NaCl
• 0.186 g/L KCl
• 2.4 g/L MgSO₄
• 5 g/L yeast extract
• Fill up with water to 1 L
• Autoclave the bottle with medium
• Per 1L of SOB, add 20mL of filter-sterilized 20% glucose solution

• 20 g/L tryptone/peptone
• 20 g/L glucose
• 10 g/L yeast extract
• Fill up with water to 1 L
• Autoclave the bottle with medium

• 20 g/L tryptone/peptone
• 20 g/L glucose
• 10 g/L yeast extract
• 20 g/L agar
• Fill up with water to 1 L
• Autoclave the bottle with medium

• 20 g/L tryptone/peptone
• 20 g/L glucose
• 10 g/L yeast extract
• 1.56 g/L histidin
• 20 g/L agar
• Fill up with water to 1 L
• Autoclave the bottle with medium

• 15 g/L agar
• Fill up with water to 800 ml
• Autoclave the bottle with medium
• Cool solution to below 60°C and add YNB, Dextrose and Biotin solutions.
• 100 ml YNB solution (13.4%)
• 100 ml dextrose (20%)
• 2 ml Biotion (0.02%)

• 242 g Tris base
• 57.1 ml Glacial acetic acid
• 37.2 g EDTA
• Fill up with distilled water to approximately 950 ml
• Measure the pH of the solution using a pH meter. Adjust the pH to approximately 8.2 with concentrated acetic acid or concentrated NaOH as needed.
• Stirring plate or magnetic stir bar
• Fill up with distilled water to 1000 ml

• 30.3 g Tris base
• 144 g glycin
• 100g SDS (Sodium Dodecyl Sulfate)
• Fill up with distilled water to approximately 950 ml
• Fill up with distilled water to 1000 ml

• Centrifuge the fragments and primers with Short Spin for approx. 10 sec.
• Dilute the fragments and primers with ddH₂O (final concentrations 50 nM for fragments and 100 µM for primers).
• Vortex thoroughly
• Short spin for approx. 10 sec.

Materials:

• Q5 2x Master Mix
• 10 µM Primer fw
• 10 µM Primer rv
• Distilled water (ddH₂O)
• DNA template
• PCR tubes/strips
• PCR machine

Procedure:

1. Preparation of PCR Reaction Mix (50 µl):
   • Mix the following components in a PCR tube to achieve a total volume of 50 µl per reaction:

   Components	Volume	Concentration
   Q5 2x Master Mix	25 µl	-
   10 µM Primer fw	2.5 µl	0.5 µM
   10 µM Primer rv	2.5 µl	0.5 µM
   ddH2O	19 µl	-
   DNA Template	1 µl	-

2. PCR Amplification (Cycling Conditions):

   Set up the PCR machine with the following cycling conditions listed in Table 2.

3. PCR Cycling Conditions (Table 2):

   Step	Temp for Fragment	Time for Fragment	Temp for Vector	Time for Vector
   Initial Denaturation	98°C	30 s	98°C	30 s
   Cycle: Denaturation	98°C	10 s	98°C	10 s
   Cycle: Annealing	70°C	30 s	70°C	30 s
   Cycle: Elongation	72°C	90 s	72°C	180 s
   Final Extension	70°C	150 s	70°C	210 s
   Hold	8°C	-	8°C	-

4. PCR Amplification:

   Run the PCR program for the specified number of cycles, 30 times for the fragments and 40 times for the vector.

5. PCR Product Analysis:

   Analyze the PCR products by running a portion of the reaction on an agarose gel for size comparison.

Materials:

• DNA samples containing the target DNA sequences
• Q5 MasterMix
• Primers specific to the target DNA sequences
• PCR tubes/strips
• PCR machine
• Agarose gel
• Gel electrophoresis equipment
• DNA ladder

Procedure:

1. DNA Extraction and Quantification:
   • Extract DNA from the respective sources and quantitate the DNA using a suitable method.
2. Primer Design:
   • Design primers specific to the target DNA sequences that will be used to amplify the linkage between the two DNA regions.
3. PCR Reaction Mix Preparation:
   • Calculate the amount of each component for a single PCR reaction based on the number of reactions you plan to perform and prepare the PCR reaction mix accordingly.
4. PCR Reaction Setup:
   • Place a PCR tube or strip into a PCR tube rack.
   • Dispense an appropriate volume of the PCR reaction mix into each PCR tube according to the calculations.
5. DNA Template Addition:
   • Add the DNA templates to the respective PCR tubes, ensuring the appropriate amount of DNA is added for each sample.
6. PCR Amplification:
   • Load the PCR tubes into the PCR machine.
   • Set up the PCR program, including denaturation, annealing, and extension temperatures and times based on the primer specifications and target DNA.
7. PCR Product Analysis:
   • After PCR amplification, analyze the PCR products by running a portion of the reaction on an agarose gel with a DNA ladder for size comparison.
   • Visualize the DNA bands under UV light using a gel documentation system.

• 0,4 g Agarose
• 50 ml 1x TAE-Puffer

1. Heat in microwave until the agarose is completely dissolved.

-Use 3,0 μl Midori-Green (Advance DNA stain) per 50 ml.

2. Allow to harden for 30 min.

For 1% Gel use 0,5g Agarose, etc.

For a larger gel-chamber and 0,8% gel: use 250 ml TAE-Puffer on 2 g Agarose

Materials:

• Agarose powder
• 1x TAE buffer
• Loading dye (6x)
• PCR product

Procedure:

1. Preparation of 0.8% w/v Agarose Gel:
   • Measure the required amount of agarose powder (w/v) for a 0.8% gel according to the desired gel size/volume.
   • Add the measured agarose powder to an appropriate volume of 1x TAE buffer in a microwave-safe flask or container.
   • Mix well to ensure the agarose is completely dissolved.
   • Microwave the mixture until the agarose is fully dissolved, but be cautious to avoid boiling over.
   • Allow the mixture to cool to around 50-60°C.
2. Addition of Loading Dye to PCR Product:
   • Add 5 µl or 10 µl of loading dye (6x) to 25 µl or 50 µl of the PCR product, respectively.
   • Mix the mixture well using a pipette to ensure thorough mixing of the PCR product and loading dye.
3. Loading PCR Product on Agarose Gel:
   • Prepare the agarose gel mold and comb to create gel pockets suitable for loading the PCR product.
   • Once the agarose mixture has cooled to around 50-60°C, pour it into the gel mold to create the gel.
   • Allow the gel to solidify by cooling at room temperature.
4. Gel Electrophoresis:
   • Carefully remove the comb from the solidified agarose gel to create wells for loading the DNA samples.
   • Place the gel in the gel electrophoresis chamber and add enough 1x TAE buffer till the fill band.
   • Load the PCR product mixed with loading dye into the wells created in the gel.
5. Gel Electrophoresis Running:
   • Run the gel at 100 V for 60 minutes.
6. DNA Fragment Visualization:
   • After electrophoresis, carefully remove the gel from the gel chamber.
   • Visualize the DNA fragments by placing the gel on a UV table and turning on the UV light.
   • Observe and document the DNA fragments based on their migration pattern and size.

Materials:

• Agarose gel containing DNA fragments
• NEB Monarch® DNA Gel Extraction Kit (or similar)
• Midori Green solution
• Sterile water
• Microcentrifuge tubes
• Microcentrifuge
• Vortex mixer
• Refrigerated centrifuge
• UV table

Procedure:

1. Preparation of the Gel:
   • Prepare an agarose gel containing DNA fragments using standard agarose gel electrophoresis procedures.
   • Stain the gel with Midori Green according to the manufacturer's instructions.
   • Visualize the DNA bands using a UV table.
2. Gel Excision:
   • Using a clean scalpel or razor blade, carefully cut out the DNA band of interest from the gel.
3. Gel Extraction Preparation:
   • Weigh the gel slice to accurately determine the amount of gel for subsequent steps.
   • Transfer the gel slice into a microcentrifuge tube.
4. Gel Dissolution:
   • Add 3-4 volumes (in μl) of Monarch Gel Dissolving Buffer to the gel slice, based on the weight of the gel slice.
   • Incubate the tube at 50°C for 5-10 minutes or until the gel slice is completely dissolved.
   • Mix the contents by inverting the tube periodically.
5. DNA Binding:
   • Place a Monarch DNA Gel Extraction Column in a collection tube.
   • Transfer the dissolved gel mixture onto the column.
   • Centrifuge at 16,000 x g for 1 minute to bind the DNA to the column.
6. Wash Steps:
   • Discard the flow-through and place the column back into the collection tube.
   • Wash the column by adding 200 μl of Monarch DNA Wash Buffer to the column.
   • Centrifuge at 16,000 x g for 1 minute. Discard the flow-through and repeat this wash step once more.
7. DNA Elution:
   • Transfer the column to a clean 1.5 ml microcentrifuge tube.
   • Add 10 μl of Monarch DNA Elution Buffer directly onto the center of the column membrane.
   • Incubate at room temperature for 1 minute.
   • Centrifuge at 16,000 x g for 1 minute to elute the purified DNA.
8. DNA Quantification:
   • Measure the concentration and purity of the purified DNA using a spectrophotometer or Nanodrop.

Materials:

• PCR reaction mixture
• NEB Monarch® PCR & DNA Cleanup Kit (or similar)
• Ethanol (95-100%)
• Sterile water
• Microcentrifuge tubes
• Microcentrifuge
• Vortex mixer

Procedure:

1. PCR Amplification:
   • Perform a standard PCR amplification according to the PCR protocol for your target DNA.
2. Preparation of PCR Products:
   • After PCR amplification, proceed with the purification process immediately or store the PCR reaction mixture at -20°C for later purification.
3. Sample Preparation for Cleanup:
   • Add 5 volumes of Monarch Binding Buffer to the PCR reaction mixture for dsDNA below 2 kb.
   • Add 2 volumes of Monarch Binding Buffer to the PCR reaction mixture for dsDNA above 2 kb.
4. DNA Binding:
   • Mix the sample thoroughly by pipetting or vortexing.
   • Transfer the mixture to a Monarch column placed in a collection tube.
   • Centrifuge at 16,000 x g for 1 minute to bind the DNA to the column.
5. Wash Steps:
   • Discard the flow-through and place the column back into the collection tube.
   • Add 200 μl of Monarch Wash Buffer to the column.
   • Centrifuge at 16,000 x g for 1 minute. Discard the flow-through and repeat this wash step once more.
6. DNA Elution:
   • Transfer the column to a clean 1.5 ml microcentrifuge tube.
   • Eluate with 10 μl of Monarch Elution Buffer by putting it directly onto the center of the column membrane.
   • Incubate at room temperature for 1 minute.
   • Centrifuge at 16,000 x g for 1 minute to elute the purified DNA.
7. DNA Quantification:
   • Measure the concentration and purity of the purified DNA using a spectrophotometer or Nanodrop.

Materials:

• DNA fragments to be assembled
• NEBuilder® HiFi DNA Assembly Master Mix
• Distilled water
• PCR machine or thermal cycler

Procedure:

1. Calculate Amounts of DNA Fragments:
   • Calculate the amount of each DNA fragment to be used in the Gibson Assembly based on their respective lengths and concentrations. Typically, equimolar amounts of fragments are used.
   • In the table below are the molar concentrations used.
cool
2. Mix DNA Fragments and Master Mix:
   • In a sterile microcentrifuge tube, combine the DNA fragments in equimolar amounts. The total DNA amount should be within the recommended range for Gibson Assembly.
   • Add an appropriate volume of NEBuilder® HiFi DNA Assembly Master Mix based on the total DNA amount. Refer to the manufacturer's instructions for the correct ratio.
3. Incubation:
   • Mix the DNA fragments and Master Mix thoroughly by gentle pipetting.
   • Incubate the reaction at the 50°C for the 15 minutes.
4. Transformation:
   • Use 3 µl of the Gibson Assembly reaction to transform into 25 µl competent E. coli cells as per standard transformation protocols.
5. Plating and Selection:
   • Plate the transformed cells on selective agar plates containing the appropriate antibiotic(.
   • Incubate the plates overnight at the 37°C.

Materials:

• BioBrick DNA parts (recipient and insert)
• Restriction enzymes (EcoRI, XbaI, SpeI, PstI)
• DNA ligase
• 10X restriction enzyme buffer
• Gel extraction kit
• Sterile water
• PCR machine
• Incubator set to 37°C

Procedure:

1. Preparation of DNA Parts:
   • Retrieve the BioBrick DNA parts (recipient and insert) from the freezer and thaw on ice.
   • Quantify the DNA concentration using a spectrophotometer or Nanodrop.
2. Digestion of DNA Parts:
   • Set up separate digestion reactions for the recipient and insert DNA parts.
   • Mix 1000 ng of each DNA part, 5 μl of 10X restriction enzyme buffer, and appropriate restriction enzymes according to RFC10 (EcoRI and XbaI for recipient, SpeI and PstI for insert).
   • Incubate the reactions at 37°C for 15 minutes and after that at 80°C for 20 minutes.
3. Gel Electrophoresis:
   • Prepare a 1% agarose gel and load the digested DNA samples alongside a DNA ladder.
   • Run the gel at 100 V for about 60 minutes.
   • Visualize the bands using a UV table.
4. Gel Extraction:
   • Cut out the gel bands corresponding to the digested DNA fragments.
   • Purify the DNA fragments using a gel extraction kit following the manufacturer's protocol.
5. Ligation:
   • Mix the purified insert DNA (SpeI-PstI digested) and the purified recipient DNA (EcoRI-XbaI digested) in a 1:1 molar ratio.
   • Add DNA ligase and ligase buffer according to the manufacturer's instructions.
   • Incubate the ligation reaction at 16°C overnight.
6. Transformation:
   • Heat-shock E. coli competent cells with the ligated DNA according to standard protocols.
   • Plate the transformed cells on appropriate selective agar plates.
7. Colony PCR and Sequencing:
   • Pick colonies from the selective agar plates and perform colony PCR using BioBrick-specific primers to confirm successful assembly.
   • Sequence the PCR products to validate the correct assembly.

Materials:

• DNA sample to be purified
• Monarch® PCR & DNA Cleanup Kit
• Microcentrifuge tubes

Procedure:

1. Binding DNA to Monarch® Columns:
   • Place a Monarch® Column in a provided collection tube.
   • Add the DNA sample (not exceeding 5 μg) to the Monarch® Column.
2. DNA Binding and Washing:
   • Add 2 volumes of Binding Buffer to the DNA sample in the Monarch® Column.
   • Mix the contents by pipetting gently.
   • Centrifuge at maximum speed for 30 seconds.
   • Discard the flow-through.
3. Washing Steps:
   • Add 700 µl of Wash Buffer to the Monarch® Column.
   • Centrifuge at maximum speed for 30 seconds.
   • Discard the flow-through.
   • Repeat the wash step once.
4. Elution of DNA:
   • Transfer the Monarch® Column to a clean microcentrifuge tube.
   • Add the provided Elution Buffer or distilled water directly to the center of the column membrane (min. 10 µl and up to 100 µl) for DNA elution.
   • Incubate at room temperature for 1 minute.
   • Centrifuge at maximum speed for 30 seconds to elute the DNA.
5. Checking DNA Concentration:
   • Measure the DNA concentration using a Nanodrop.

Materials:

• LB (Luria-Bertani) or TB (Terrific Broth) medium
• Ampicillin solution (5 µL per culture tube)
• Sterile pipette tips
• Bacterial culture plate

Procedure:

1. Preparation of Culture Medium:
   • Depending on the purpose, select either LB or TB medium for the bacterial culture.
   • Measure 5 mL of the chosen medium and add it to a sterile culture tube.
2. Addition of Ampicillin:
   • Measure 5 µL of ampicillin using a micropipette and add it to the culture tube containing the medium. Ampicillin is added to promote the growth of bacteria harboring a plasmid with ampicillin resistance.
3. Colony Selection and Inoculation:
   • Using a sterile pipette tip, carefully pick a single bacterial colony from the culture plate that you want to inoculate.
   • Transfer the picked colony into the culture tube containing the medium and ampicillin.
4. Incubation:
   • Immerse the pipette tip with the picked colony into the medium-antibiotic mixture in the culture tube.
   • Incubate the culture tube at 37°C to promote bacterial growth and propagation. The culture can be incubated in an appropriate incubator or shaker.

Materials:

• Bacterial culture in LB medium (10 mL)
• LB medium
• CaCl2 (50 mM)
• CC Buffer
• Liquid Nitrogen

Procedure:

1. Overculture in LB Medium:

   Prepare an overculture by incubating the bacterial culture in 10 mL of LB medium at the appropriate temperature and conditions until reaching the desired growth phase.

2. Inoculation and Growth:

   • Inoculate 50 mL of fresh LB medium with the overculture to achieve an initial OD600 of 0.1.
   • Incubate the culture at 37°C until the OD600 reaches approximately 0.6.

3. Centrifugation and Supernatant Discard:

   • Centrifuge the bacterial culture at 5000 rpm and 4°C for 5 minutes.
   • Discard the supernatant.

4. Resuspension in CaCl2 (50 mM):

   • Resuspend the bacterial pellet in 25 mL of CaCl2 (50 mM).
   • Centrifuge at 5000 rpm and 4°C for 5 minutes.
   • Discard the supernatant.

5. Resuspension in CC Buffer:

   • Resuspend the bacterial pellet in 10 mL of CC Buffer.
   • Centrifuge at 5000 rpm and 4°C for 5 minutes.
   • Discard the supernatant.

6. Division into Aliquots:

   • Resuspend the bacterial pellet in 6 mL of CC Buffer.
   • Divide the suspension into 200 µl aliquots.
   • Freeze each aliquot immediately in liquid nitrogen.

Materials:

• Pichia pastoris cells (grown to mid-log phase)
• 1 M Sorbitol solution
• Lithium chloride (LiCl) solution (0.1 M)
• Ice
• Centrifuge
• Sterile microcentrifuge tubes
• Luria-Bertani (LB) agar plates

Procedure:

1. Prepare Pichia pastoris Culture:

Grow Pichia pastoris cells in YPD (Yeast extract Peptone Dextrose) medium to mid-log phase (OD600 ≈ 0.6-1.0) by incubating at 30°C with shaking at 220 rpm.

2. Harvest the Cells:

• Transfer the culture to a sterile centrifuge tube and centrifuge at 3000 rpm for 5 minutes at 4°C.
• Discard the supernatant and resuspend the pellet gently in an equal volume of 1 M sorbitol.

3. Wash and Resuspend the Cells:

• Centrifuge the cells again at 3000 rpm for 5 minutes at 4°C.
• Discard the supernatant and resuspend the pellet gently in an equal volume of ice-cold 1 M sorbitol.

4. Centrifuge and Resuspend in LiCl:

• Centrifuge the cells at 3000 rpm for 5 minutes at 4°C.
• Discard the supernatant and resuspend the pellet in an equal volume of ice-cold 0.1 M LiCl.
• Incubate the cells on ice for 1 hour.

5. Centrifuge and Wash with LiCl:

• Centrifuge the cells again at 3000 rpm for 5 minutes at 4°C.
• Discard the supernatant and resuspend the pellet in 1 mL of ice-cold 0.1 M LiCl.
• Centrifuge the cells at 3000 rpm for 5 minutes at 4°C.
• Discard the supernatant and resuspend the pellet in a small volume (e.g., 100-200 μL) of ice-cold 0.1 M LiCl.

Materials:

• Competent cells
• Plasmid DNA (positive control)
• Gibson construct (3 µl)
• BioBrick construct (2 µl)
• LB medium
• LB agar culture plates with appropriate antibiotic
• Ice
• Thermocycler
• Centrifuge

Procedure:

1. Thawing Competent Cells:
   • Thaw 25 µL of competent cells on ice.
2. Pre-incubation of Competent Cells:
   • Leave the cells on ice for 25 to 30 minutes.
3. DNA Addition:
   • Add the following to the competent cells while keeping the tube on ice:
     • 1 µL of plasmid DNA (positive control)
     • 3 µL of Gibson construct
     • 2 µL of BioBrick construct
4. Incubation on Ice:
   • Incubate the cells with DNA for 30 minutes on ice.
5. Heat Shock Treatment:
   • Place the tube with the cells and DNA in a thermocycler at 42°C for 45 seconds.
6. Cooling on Ice:
   • Place the cells on ice for 1 minute to cool down.
7. Addition of LB Medium:
   • Add 450 µL of LB medium to the cells.
8. Incubation:
   • Incubate the cells for at least 60 minutes at 37°C.
9. Centrifugation (for Gibson Assembly):
   • Centrifuge the cells with Gibson Assembly constructs at 13,000 rpm for 1 minute.
   • Discard 350 µL of the supernatant, resuspend the pellet, and retain the remainder.
10. Plating of Cells:
    • Plate out 100 μL of the cell suspension directly onto LB agar culture plates with the appropriate antibiotic.
11. Overnight Incubation:
    • Incubate the culture plates overnight at 37°C.

Objective:

The objective of this protocol is to quickly analyze Gibson-assembled plasmids.

Materials:

• Cell samples from the backup plates
• Sterile ddH2O (deionized water)
• 2x NEB solution (NSEB)
• 25% Ficoll
• Agarose gel

Procedure:

1. Cell Preparation:
   • Extract a small amount of cells from the backup plates and dissolve them in 15 µl of sterile ddH2O.
2. Master Mix Preparation:
   • Prepare a Master Mix using 2x NSEB solution.
3. Incubation:
   • Incubate the cell samples with the Master Mix for 1 hour at 98°C.
4. Ficoll Mixing:
   • Mix the incubated cells with 25% Ficoll.
5. Gel Application:
   • Apply the mixture onto an agarose gel for analysis.

Materials:

• Bacterial colonies
• NEB Q5 MasterMix
• Primers specific to the target DNA
• Sterile water
• PCR tubes/strips
• PCR machine
• Agarose gel
• Gel electrophoresis equipment
• DNA ladder

Procedure:

1. Colony Selection:
   • Select bacterial colonies from a freshly streaked agar plate that contains the desired DNA fragment.
2. Preparation of PCR Reaction Mix:
   • Prepare a master mix for the PCR reaction by combining the following components in a sterile microcentrifuge tube:
     • NEB Q5 MasterMix
     • Primers (forward and reverse) specific to the target DNA
     • Sterile water
3. PCR Reaction Setup:
   • Place a PCR tube or strip into a PCR tube rack.
   • Dispense an appropriate volume of the PCR reaction mix (calculated for each reaction) into each PCR tube.
4. Collection of Bacterial Cells:
   • Using a sterile pipette tip, pick a small amount of a bacterial colony from the streaked agar plate.
5. Inoculation into PCR Reaction Mix:
   • Transfer the picked colony directly into the PCR tube containing the reaction mix. Mix gently by pipetting up and down.
6. PCR Amplification:
   • Load the PCR tubes into the PCR machine.
   • Set up the PCR program, including denaturation, annealing, and extension temperatures and times based on the primer specifications and target DNA.
7. PCR Program:
   • Perform PCR amplification using the following program as a general guideline:
     • Initial denaturation: 98°C for 30 seconds
     • Denaturation: 98°C for 10 seconds
     • Annealing: Optimal temperature for primer pair (e.g., 55-65°C) for 20 seconds
     • Extension: 72°C for appropriate time per kb of expected PCR product (e.g., 30 seconds per kb)
     • Final extension: 72°C for 2-5 minutes
8. PCR Product Analysis:
   • After PCR amplification, analyze the PCR products by running a portion of the reaction on an agarose gel with a DNA ladder for size comparison.
   • Visualize the DNA bands under UV light using a gel documentation system or UV table.

Materials:

• Bacterial colonies
• Sterile water
• 20 µl of 20 mM NaOH
• PCR master mix
• PCR tubes/strips
• PCR machine
• Agarose gel
• Gel electrophoresis equipment
• DNA ladder

Procedure:

1. Colony Selection and Lysis:
   • Select bacterial colonies from an agar plate.
   • In a sterile microcentrifuge tube, add a single colony from each selected bacterial colony to 20 µl of 20 mM NaOH.
   • Mix gently to ensure thorough mixing of cells with NaOH.
   • Incubate the mixture at 98°C for 45 minutes for cell lysis.
2. Neutralization:
   • After the incubation, allow the lysed cells to cool down to room temperature.
3. PCR Setup:
   • Add 1-2 µl of the lysed cell suspension to the PCR tubes containing the PCR master mix.
4. PCR Amplification:
   • Load the PCR tubes into the PCR machine.
   • Set up the PCR program, including denaturation, annealing, and extension temperatures and times based on the primer specifications and target DNA.
5. PCR Product Analysis:
   • After PCR amplification, analyze the PCR products by running a portion of the reaction on an agarose gel with a DNA ladder for size comparison.
   • Visualize the DNA bands under UV light using a gel documentation system or UV table.

Materials:

• E. coli culture containing the plasmid of interest
• innuPREP Plasmid Mini Kit 2.0 (Jena Analytik)
• Lysis buffer
• Neutralization buffer
• Wash buffer (I and II)
• Elution buffer
• Microcentrifuge tubes
• Microcentrifuge
• Centrifuge

Procedure:

1. Harvesting Bacterial Culture:
   • Inoculate a suitable volume of E. coli culture containing the plasmid of interest and grow it to the desired cell density.
2. Pellet Cells:
   • Transfer 1.5–2 mL of the bacterial culture into a microcentrifuge tube.
   • Centrifuge at 8,000 × g for 2 minutes to pellet the bacterial cells.
3. Resuspension of Cell Pellet:
   • Discard the supernatant carefully.
   • Resuspend the cell pellet in 250 µL of lysis buffer by gentle vortexing or pipetting.
   • Ensure thorough mixing to achieve a homogenous cell lysate.
4. Lysis of Cells:
   • Incubate the resuspended cells in lysis buffer at room temperature for 10 minutes.
5. Neutralization:
   • Add 300 µL of neutralization buffer and mix gently by inverting the tube several times.
   • Incubate at room temperature for 5 minutes.
6. Binding Plasmid DNA:
   • Load the lysate onto the innuPREP spin filter placed in a collection tube.
   • Centrifuge at 13,000 × g for 1 minute.
   • Discard the flow-through.
7. Washing:
   • Add 500 µL of wash buffer I to the spin filter.
   • Centrifuge at 13,000 × g for 1 minute.
   • Discard the flow-through.
   • Add 700 µL of wash buffer II to the spin filter.
   • Centrifuge at 13,000 × g for 1 minute.
   • Discard the flow-through.
8. Elution of Plasmid DNA:
   • Place the spin filter in a clean microcentrifuge tube.
   • Add 50 µL of elution buffer to the center of the spin filter.
   • Incubate at room temperature for 2 minutes.
   • Centrifuge at 13,000 × g for 1 minute to elute the plasmid DNA.
9. Quantification and Storage:
   • Quantify the isolated plasmid DNA using a Nanodrop.

• DNA samples in a concentration of at least 50 ng/µl
• Barcode labels

Procedure:

1. Prepare DNA Samples:
   • Ensure you have DNA samples available, each with a concentration of at least 50 ng/µl.
2. Labeling with Barcode:
   • Affix a barcode label to each tube containing the DNA sample.
3. Volume Measurement:
   • Measure and pipette 5 µL of the DNA sample into each labeled tube.
   • Pipette 5 µL of the 5 µM primer into each labeled tube.
   • Send the labeled tube.

Materials:

• Agarose gel with separated DNA fragments
• Midori Green DNA staining solution
• Gel electrophoresis chamber
• UV table

Procedure:

1. Gel Preparation:
   • Perform agarose gel electrophoresis to separate DNA fragments based on size and charge.
2. Preparing Midori Green Staining Solution:
   • Dilute Midori Green DNA staining solution as per the manufacturer's instructions to the desired concentration for staining.
3. Poststaining:
   • After completing the gel electrophoresis, remove the gel from the gel electrophoresis chamber.
4. Midori Green Staining:
   • Submerge the gel in the prepared Midori Green staining solution, ensuring the gel is completely covered.
5. Staining Duration:
   • Allow the gel to stain for 20-30 minutes. The staining time can be adjusted based on the intensity of staining required.
   • Wash the gel twice for 30 minutes with water.
6. Visualization:
   • After staining, visualize the DNA bands under a UV table.

Materials:

• Agarose gel with separated DNA fragments
• GelRed nucleic acid stain
• Gel electrophoresis chamber
• UV table

Procedure:

1. Gel Preparation:
   • Perform agarose gel electrophoresis to separate DNA fragments based on size and charge.
2. Preparing GelRed Staining Solution:
   • Dilute the GelRed nucleic acid stain according to the manufacturer's instructions to the desired concentration for staining.
3. Poststaining:
   • After completing the gel electrophoresis, remove the gel from the gel electrophoresis chamber.
4. GelRed Staining:
   • Submerge the gel in the prepared GelRed staining solution, ensuring the gel is completely covered.
5. Staining Duration:
   • Allow the gel to stain for 15 minutes. The staining time can be adjusted based on the intensity of staining required.
   • Wash the gel twice for 30 minutes with water.
6. Visualization:
   • After staining, visualize the DNA bands under a UV transilluminator. GelRed fluoresces under UV light, allowing for easy visualization of DNA bands.

Materials:

• Plasmid containing the protein of interest
• E. coli strain BW25113
• Antibiotic selection plates (ampicillin)
• TB medium (Terrific Broth)
• TB salt
• 10% arabinose
• Spectrophotometer (measuring OD at 600 nm)
• Shaking incubator

Procedure:

1. Plasmid Transformation and Overnight Culture:
   • Transform the plasmid containing the protein of interest into BW25113 E. coli cells.
   • Plate the transformed cells on antibiotic selection plates (ampicillin) and incubate overnight at 37°C.
2. Preparing Overnight Culture:
   • Pick a single colony from the antibiotic selection plate and inoculate it into 5 ml of TB medium containing TB salt and ampicillin.
   • Incubate the culture overnight at 37°C with shaking.
3. Scaling Up the Culture:
   • Transfer the entire overnight culture into 500 ml of TB medium in a larger flask.
4. Incubation and Growth Monitoring:
   • Incubate the culture at 37°C with shaking.
   • Continuously measure the optical density (OD) at 600 nm at regular intervals and record the values.
5. Induction:
   • When the OD value is between 0.7 and 0.9, induce protein expression.
   • Add 10 ml of 10% arabinose to reach the desired concentration for induction. Adjust the total volume accordingly.
6. Expressing Protein:
   • Continue incubation after induction for the desired period at the designated temperature:
   • 37°C or 30°C.
7. Protein Harvesting:
   • Harvest the cells by centrifugation at an 14000 rpm and 15 minutes to obtain the cell pellet.

Materials:

• Bovine Serum Albumin (BSA)
• Distilled water (ddH2O)
• Bradford reagent
• Cuvettes
• Spectrophotometer (set to 595 nm)
• Parafilm

Procedure:

Preparation of BSA Standard Curve:

1. Prepare BSA Standard Solutions:
   • Dissolve 1 mg of BSA in 1 mL of ddH2O to prepare a 1 mg/mL BSA standard solution.
2. Prepare Triplicate Standard Samples:

   BSA [µg]	BSA Standard [µL]	H2O [µL]
   0	0	100
   2	2	98
   5	5	95
   7	7	93
   10	10	90
   15	15	85
   20	20	80
   25	25	75

3. Prepare Cuvettes:
   • Pipette the triplicate standard samples directly into cuvettes as per the table.
4. Spectrophotometer Setup:
   • Set the spectrophotometer to measure at 595 nm.
5. Add Bradford Reagent:
   • Add the Bradford reagent to each cuvette and gently invert the samples using Parafilm.
   • Measure the absorbance at exactly 5 minutes.
6. Record Absorbance:
   • Note the absorbance values at 595 nm for each standard sample.

Preparing Unknown Samples:

1. Sample Preparation:
   • Use 100 µL of the diluted sample and add 900 µL of Bradford reagent.
2. Measurement:
   • Measure the absorbance at 595 nm following steps 3-5 of the BSA standard curve preparation.
3. Adjust Sample Volume:
   • Adjust the used sample volume as needed to stay within the linear range of the standard samples (2-25 µg/mL).

Materials:

• Protein samples
• 2x SDS loading buffer
• Marker
• SDS-PAGE gel
• SDS running buffer
• Coomassie staining solution
• 20% acetic acid
• Distilled water (ddH2O)
• Electrophoresis apparatus
• Power supply

Procedure:

1. Sample Preparation:
   • Apply approximately 10 µg of protein per sample. If needed, dilute the sample to achieve the desired protein concentration.
2. Mixing with SDS Loading Buffer:
   • Mix 7.5 µL of each sample with 7.5 µL of 2x SDS loading buffer.
3. Boiling:
   • Boil the sample-buffer mixture at 95°C for 10 minutes.
4. Applying Marker and Samples:
   • Apply 5 µL of the marker and 15 µL of each sample mixed with the loading buffer onto the SDS-PAGE gel.
5. Running SDS-PAGE:
   • Run the SDS-PAGE at 100 V and 200 mA for 1.5 hours.
6. Staining with Coomassie:
   • Stain the gel with Coomassie staining solution for 40 minutes. Ensure the gel is completely covered.
7. Destaining:
   • Destain the gel with 20% acetic acid for 60 minutes.
   • Subsequently, destain the gel with distilled water (ddH2O) overnight.
8. Visualization:
   • Visualize the protein bands on the gel.
   • Document the results using an appropriate imaging system.

Protocol for Isolating Genomic DNA from Pichia pastoris

Materials:

• Pichia pastoris cells (grown culture)
• TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
• Lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 1% SDS, pH 8.0)
• Phenol:chloroform:isoamyl alcohol (25:24:1)
• Chloroform:isoamyl alcohol (24:1)
• Ethanol (ice-cold)
• Isopropanol (ice-cold)
• 70% ethanol (ice-cold)
• Nuclease-free water

Procedure:

1. Cell Lysis:

• Harvest Pichia pastoris cells and wash with TE buffer.
• Resuspend the cells in lysis buffer and incubate at 65°C for 30-60 minutes.

2. DNA Extraction:

• Extract genomic DNA using phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuge to separate phases.
• Transfer the aqueous phase to a new tube.
• Precipitate DNA with ice-cold ethanol or isopropanol and centrifuge at maximum speed for 10-15 minutes.

3. DNA Wash:

• Discard the supernatant and wash the DNA pellet with 70% ethanol.
• Centrifuge at maximum speed for 5 minutes and carefully remove the ethanol.

4. DNA Resuspension:

• Allow the DNA pellet to air dry briefly and then resuspend in TE buffer or nuclease-free water.
• Incubate at 65°C for 10-15 minutes to aid in resuspension.

5. DNA Quantification:

• Quantify the isolated genomic DNA using a spectrophotometer.

Materials:

• n-Hexane
• tert-Butanol
• Lauric Acid
• Candida Antarctica Lipase B (CALB) immobilized on beads
• Glycerol
• Orbital shaker
• Hot water (80°C)
• Volumetric flasks
• Ice bath
• Whatman filter paper
• Glycerol enzyme assay kit (Genaro)
• Spectrophotometer (measuring at 340 nm)

Procedure:

1. Preparation of Reaction Mixture:
   • In a shaking flask (200 ml end volume) with a cap, prepare a mixture containing:
     • 5.21 g (0.13 mol) lauric acid dissolved in hexane
     • 2.3944 g (0.13 mol) glycerol
     • Immobilized lipase (160 mg, do not dissolve in water)
     • Add hexane (or 1:1 hexane/tert-butanol) to make a total volume of 200 ml.
   • Prepare a control without enzyme.
2. Incubation:
   • Incubate the mixture using an orbital shaker at 300 rpm/min at two different temperatures: 40°C and 65°C.
3. Sample Collection:
   • Take samples (1.5 ml) every 30 minutes and keep them on ice until further measurement or fat extraction.
4. Hot Water Extraction for Fat Separation:
   • For samples with high fat content:
     • Weigh the samples into a volumetric flask.
     • Extract with hot water (80°C).
     • Allow the sample solution to cool for fat separation (e.g., 15 min in an ice bath).
     • Fill the volumetric flask up to the mark with water.
     • Filter the aqueous solution before testing using Whatman filter paper.
5. Sample Dilution:
   • Dilute the samples 1:100 at the beginning of the measurements to achieve a linear range of 0.04 to 0.4 g/l.
6. Spectrophotometric Measurement:
   • Measure the samples at 340 nm using a spectrophotometer.
7. Glycerol Enzyme Assay:
   • Perform the glycerol enzyme assay using the Glycerol enzyme assay kit, following the provided manual.