1. To a microcentrifuge tube, add:

1. To a microcentrifuge tube, add:
• Digested vector DNA (in appropriate ratio)
• Digested insert DNA (in appropriate ratio)
• 1/10 of total final volume of aliquoted 10X T4 DNA ligase buffer
• 1μl of T4 DNA ligase (1U/μl)
• ddH2O to final volume
2. Incubate at room temperature for 2 hours
3. Transform chemically competent cells with the ligation product.
4. Leave remaining ligation product at room temperature overnight, and transform again the following day.
5. Store remaining product at -20°C

1. Add 10μl of ddH2O to the desired well of the distribution kit plate
2. .Pipette up and down 3-5 times (until solution becomes red)
3. Incubate at room temperature for 10 minutes
4. Transform cells with 1μl of rehydrated DNA as per transformation protocol

1. Combine in a 0.2ml microcentrifuge tube:
• 5μl of NEB 10X standard Taq buffer (final concentration = 1X)
• 0.25μl of NEB Taq (final concentration = 1.25U/50μl)
• 1μl of 10μM forward primer (final concentration = 0.2μM)
• 1μl of 10μM reverse primer (final concentration = 0.2μM)
• 1pg to 1ng of template plasmid DNA (final concentration < 1000ng/μl)
• 1μl of 10mM Kapa dNTPs (final concentration = 200μM)
• ddH2O to 50μl
2. Vortex 2 to 3 seconds to mix, then centrifuge briefly to settle
3. Place in thermocycler and select or set up the appropriate program as follows:
• Initial denaturation → 95°C for 5 minutes
• Repeat 30x:
• Denaturation → 95°C for 30 seconds
• Annealing → Tm - 5°C (45 to 68°C) for 15 to 60 seconds
• Extension → 68°C for 1 minute per kilobase
• Final extension → 68°C for 5 minutes
• Final extension → 68°C for 5 minutes
• Hold at 4°C

1. Add 10μl of ddH2O to the desired well of the distribution kit plate
2. .Pipette up and down 3-5 times (until solution becomes red)
3. Incubate at room temperature for 10 minutes
4. Transform cells with 1μl of rehydrated DNA as per transformation protocol

1. To a microcentrifuge tube, add:
• Required amount of DNA to be digested (can use NEBioCalculator to determine amount if performing a ligation with digested DNA)
• 1/10 final total volume of appropriate 10X buffer
• 1μl of each restriction enzyme (diluted)
• ddH2O to final volume
2. Incubate at 37°C (or other optimal temperature for enzyme activity) for 30 minutes to 3 hours (be careful of star activity)
3. Heat inactivate restriction enzymes at 82°C for 20 minutes
4. For digest confirmations, run on regular agarose gel. For subsequent ligation, run on LMP gel and excise or gel-extract. Otherwise, store at -20°C

1. Add ≤ 5μl (≤ 1/10 of the cell aliquot amount) of the DNA sample to a chemically competent cell aliquot. Mix by pipetting gently, then incubate on ice for 30 to 45 minutes.
2. Heat shock at 42°C for 1 minute
3. Incubate on ice for 5 minutes
4. Add 250μl of plain LB or SOC media aseptically, then incubate for 30 to 90 minutes at 37°C, shaking. *If resistance is Kan, must incubate for at least 1 hour
5. Plate 100μl (if big plates) or 50μl (if small or half plates) aseptically
6. Incubate plate at 37°C overnight or until growth is observed

*If transformation fails:

• Spin down transformed cells for 5 minutes to pellet
• Resuspend pellet in 100μl of media
• Plate and incubate

1. Culture overnight culture (O/N) in 2ml LB at 28°C, shaking
2. Subculture (1:50) by adding 1ml O/N culture to 50ml LB with 10mM MgSO4 (500μl of 1M MgSO4) and 1mM KCl (50μl of 1M KCl)
3. Shake at 28°C to OD600 = 0.3 to 0.4
4. Chill on ice for at least 10 minutes
5. Put into 50ml pre-chilled Falcon tube and centrifuge at 2500g for 8 minutes at 4°C (3450rpm in Allegra X-12 centrifuge)
6. Resuspend in 10ml ice-cold 100mM CaCl2, gently mix on ice, then ice for at least 10 minutes
7. Centrifuge at 2500g for 8 minutes at 4°C
8. Resuspend in 500μl 100mM CaCl2 with 10% glycerol on ice, then incubate on ice for 10 minutes
9. Aliquot 50μl into pre-chilled 1.5ml microcentrifuge tubes on ice. Store at -80°C
10. Perform a test transformation with a reporter gene (i.e RFP)

1. Create cPCR mastermix as follows:
• 20μL 10X Taq Buffer
• 4μL 10μM VF2 primer (or other forward primer)
• 4μL 10μM VR primer (or other reverse primer)
• 4μL 10mM dNTPs
• 1μL Taq Polymerase
• 127μL ddH2O

*Each mastermix aliquot is enough to run 10 cPCR reactions/screen 10 colonies

2. Add 4μL of ddH2O to each PCR tube or well (of a 96-well plate)
3. Using pipette tips or sterile toothpicks, touch an individual labelled colony, swirl in the ddH2O, and then streak on masterplate. Repeat for as many colonies as desired.
4. Add 16μL of cPCR mastermix to each tube/well
5. Place in thermocycler and select or set up the appropriate program as follows:
• Initial denaturation → 95°C for 5 minutes
• Repeat 30x:
• Denaturation → 95°C for 30 seconds
• Annealing → Tm - 5°C (For VF2/VR primers, 53°C) for 30 seconds
• Extension → 68°C for 1 minute per kilobase
• Final extension → 68°C for 5 minutes
• Final extension → 68°C for 5 minutes
• Hold at 4°C

1. Add 500μl of 50% glycerol to a 1.5ml tube aseptically
2. Add 500μl of 50% of overnight culture to the tube aseptically. Mix gently
3. Store at -80°C

1. Mix into 1L of dH2O:
• 10g of Tryptone (1%)
• 5g of yeast extract (0.5%)
• 10g NaCl (1%)
• 15g Agar (1.5%)
2. Autoclave and cool
3. Add necessary antibiotics and mix thoroughly
• 1ml/L Ampicillin (stock = 100mg/ml, final = 100μg/ml)
• 1ml/L Kanamycin (stock = 50 mg/ml, final = 50μg/ml)
• 0.5ml/L Chloramphenicol (stock = 50 mg/mlEtOH, final = 25μg/ml)
4. Pour into plates aseptically, swirl to remove bubbles, and let set. Store in fridge upside-down.

1. Mix into 1L of dH2O:
• 10g of Tryptone (1%)
• 5g of yeast extract (0.5%)
• 10g NaCl (1%)
2. Autoclave and cool
3. Add necessary antibiotics and mix thoroughly
• 1ml/L Ampicillin (stock = 100mg/ml, final = 100μg/ml)
• 1ml/L Kanamycin (stock = 50 mg/ml, final = 50μg/ml)
• 0.5ml/L Chloramphenicol (stock = 50 mg/mlEtOH, final = 25μg/ml)
4. Pour into plates aseptically, swirl to remove bubbles, and let set. Store in fridge upside-down.
5. Aliquot as necessary

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Reagents:

• 6.7g Bactoyeast Nitrogen base without amino acids, with ammonium sulphate
• 20g sugar (typically glucose)
• 2.0mL 1% L-tryptophan
• 2.0mL 1% L-methionine
• 2.0mL 1% L-arginine
• 2.0mL 1% L-leucine
• 2.0mL 1% L-lysin
• 10mL 0.2% Adenine sulfate
• 10mL 0.2% Uracil

1. In 500mL of ddH2O dissolve:
• Bactoyeast nitrogen base (TNB, final concentration= 0.67% w/v)
• Sugar (final concetration= 2% w/v)
2. Under aseptic conditions add:
• Amino acids from AUTOCLAVED 1% solutions
• Bases from AUTOCLAVED 0.2% solutions

*Add the amino acids and bases in the oder listed above to fill the desired auxotrophies. For example, to make SD-Ura (media lacking uracil), add all the amino acids and adenine, but not uracil.

3. Bring volume to 1000mL with ddH2O, accounting for the volume(s) of amino acid and base solutions added.
4. FOR PLATES: Add agar to final concentration of 2% (i.e 20g in 1L of media)

You can either:

• Resuspend cells from a fresh plate in 1mL sterile 15% glycerol
• Add 600uL of log phase culture (overnight culture) to 400uL of sterile 50% glycerol- vortex the culture first.

Work under aseptic conditions and store the tubes in the -80ºC freezer

helloo

Solution Preparation:

1. Lithium Acetate (LiAc) Solution: 0.1M, pH 6.0
• 5.1g LiAc for 500mL
• Adujst to pH 6.0 with 10% acetic acid
• Autoclave and store in the fridge
2. Polyethylene Glycol (PEG) Solution: 40% PEG 4000 in 0.1M LiAc, pH 6.0
• Add 50mL 0.1M LiAc to 40g PEG 4000
• Adjust to pH 6.0 with 10% acetic acid
• Adjust volume to 100mL with LiAc solution
• Store in the fridge
3. DNA Carrier: 5mg/mL fish DNA (salmon sperm)
   • 50mg DNA sodium salt/fish DNA
   • Dissolve in 10mL sterile TE buffer
   • Aliquot samples and store in -20ºC freezer

Part I: Preparation of Competent Cells

1. Spread the strain that you want to transform on a YPD plate (Grow at 28°C for 16-24 hours)
2. Put a loop of cells into 1ml of TE in sterile Eppendorf tubes
• Prep for 5 transformations- i.e. 5 loops in 5 ml of TE
3. Centrifuge 1 min at 10 000 rpm and remove supernatant
4. Resuspend cell gently in 600µL of 0.1M LiAc pH 6.0
• Prep for 5 transformations- i.e. 3mL
5. Place in 28°C water bath without agitation for 1 hour
6. Centrifuge for 2 min at 3000 rpm and remove supernatant
7. Gently resuspend cells in 60µL of 0.1M LiAc pH 6.0
   • Prep for 5 transformations- i.e. 300µL

Part II: Transformation

1. In a new eppendorf tube, mix together the following with a pipette:
• 3-5μl of carrier DNA
• 2-5µL your desired DNA
• 40µL competent cells
2. Incubate for 15 min in 28°C water bath without agitation
3. Add 350µL PEG solution
4. Incubate for 1 h in 28°C water bath without agitation
5. Thermal shock the cells for 10 min at 39°C
6. Add 600µL 0.1M LiAc pH 6.0
7. Spread 200μl (or more) per plate containing the adequate selection medium

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.