Agarose Gel

Materials

• 1X TAE
• Agarose
• Gel Red
• DNA samples
• 6X loading dye
• Nuclease-free water

Procedure

1. Prepare 1.2% agarose gel for small fragments and 3% agarose gel for large fragments
2. Mix 50 mL 1X TAE and 0.6 g (1.2%) or 1.5 g (3%) agarose
3. Melt in microwave until agarose has melted (about 50 seconds)
4. Add 1.3 μL (1.2%) or 1.5 μL (3%) Gel Red
5. Pour solution into agarose gel mold with comb
6. Let set for 20 minutes or until solid
7. Place gel in 1X TAE and remove comb
8. Load samples of 200 ng (or 2 μL) DNA mixed with 2 μL 6X loading dye and nuclease-free water up to 12 μL
9. Run gel at 100-120 Volts for 40-50 minutes (1.2%) or 80 Volts for 2 hours (3%)
10. Take a picture of the gel at the UV detector

Competent Cell Preparation
Based on Open Wet Ware Protocol

Materials

• Bacterial overnight liquid culture
• Lysogeny broth (LB) medium
• CaCl2 solution, ice cold: 60 mM CaCl2, 15% glycerol, 10 mM PIPES, pH 7, filter sterilize and store at room temperature

Procedure

1. Subculture overnight culture 1:100 in LB medium
2. Incubate at 37°C with shaking until culture reaches an OD600 of 0.375
3. Aliquot 20 mL if the culture into chilled 50 mL tubes
4. Leave tubes on ice for 5 – 10 minutes
5. Centrifuge cells at 1600 g for 7 minutes at 4°C
6. Discard supernatant and resuspend pellet in 4 mL ice cold CaCl2 solution
7. Centrifuge cells at 1100 g for 5 minutes at 4°C
8. Discard supernatant and resuspend pellet in 4 mL ice cold CaCl2 solution
9. Keep on ice for 30 minutes
10. Centrifuge cells at 1100 g for 5 minutes at 4°C
11. Discard supernatant and resuspend pellet in 800 μL ice cold CaCl2 solution
12. Aliquot 100 μL of this suspension into microcentrifuge tubes
13. Freeze in liquid nitrogen and store at -80°C

Dephosphorylation of 5'-ends of DNA using Antarctic Phosphatase
Based on NEB Protocol

Materials

• 1-5 µg cut DNA
• 10X Antarctic Phosphatase Reaction Buffer
• Antarctic Phosphatase

Procedure

1. Add 1/10 volume of 10X Antarctic Phosphatase Reaction Buffer to 1-5 µg of DNA cut with any restriction endonuclease in any buffer.
2. Add 1 µl of Antarctic Phosphatase (5 units) and mix
3. Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions
4. Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 70°C
5. Proceed with ligation

Gel extraction
With QIAquick Gel Extraction Kit (QIAGEN)

Materials

• Agarose gel
• QIAquick Gel Extraction Kit

Procedure

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg = 100µL). For >2% agarose gels, add 6 volumes Buffer QG
3. Incubate at 50ºC for 10 minutes (or until the gel slice has completely dissolved). Vortex the tube every 2-3 minutes to help dissolve the gel
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10µL 3M sodium acetate, pH5.0, and mix. The color of the mixture will turn yellow
5. Add 1 gel volume of isopropanol to the sample and mix
6. Place a QIAquick spin column in a provided 2 mL collection tube
7. To bind DNA, apply the sample to QIAquick column and centrifuge for 1 min. Discard the flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 µL, load and spin again
8. To wash, add 0.75 mL Buffer PE to QIAquick column and centrifuge for 1 min. Discard the flow-through and place the QIAquick column back into the same tube
9. Centrifuge the QIAquick column once more in the provided 2 ML collection tube for 1 min at 13'000 rpm to remove residual buffer
10. Place QIAquick column into a clean 1.5 mL microcentrifuge tube
11. To elute DNA, add 50 µL Buffer EB or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.

Gibson Assembly
Based on NEB Gibson Assembly Protocol

Materials

• DNA fragments
• 2X Gibson Assembly Mater Mix (NEB)
• 2X NEBuilder Positive Control (NEB)
• Deionized water

Procedure

1. Set up following reactions on ice, adding Gibson Assembly Master Mix last:

Component	2 – 3 Fragments Assembly	4 – 6 Fragments Assembly	Positive Control
Total Amount of Fragments	0.02 – 0.5 pmols	0.2 – 1 pmols	10 μL
2X Gibson Assembly Master Mix	10 μL	10 μL	10 μL
Deionized water	to 20 μL	to 20 μL	0 μL

Optimized efficiency for 50 – 100 ng of vectors and 2 – 3 fold of excess inserts

2. Incubate samples at 50°C for 15 minutes (2 – 3 fragments) or for 60 minutes (4 – 6 fragments)
3. Store samples on ice or at -20°C until transformation
4. Transform competent cells following the Transformation Protocol

Glycerol Stock

Materials

• 50% Glycerol
• Bacterial overnight liquid culture
• Liquid Nitrogen

Procedure

1. Mix 0.5 mL 50% glycerol and 0.5 mL bacterial culture
2. Freeze in liquid nitrogen and store at -80°C

Lysogeny Broth (LB) Medium

Materials

• Tryptone
• Yeast extract
• NaCl
• Deionized water
• NaOH
• If necessary: antibiotics

Procedure (for 1 L)

1. Dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl in 950 mL deionized water
2. Adjust pH to 7 using 1 M NaOH and bring volume to 1 L
3. Autoclave
4. If necessary: let medium cool to 55°C and add atibiotic
5. Store at room temperature

Lysogeny Broth (LB) Agar Plates

Materials

• Tryptone
• Yeast extract
• NaCl
• Deionized water
• NaOH
• Agar
• If necessary: antibiotics
• Petri dishes

Procedure (for 1 L, ie. about 50 plates)

1. Dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl in 950 mL deionized water
2. Adjust pH to 7 using 1 M NaOH and bring volume to 1 L
3. Add 15 g agar
4. Autoclave
5. If necessary: let medium cool to 55°C and add atibiotic
6. Pour into petri dishes (about 20 mL per dish) and let set
7. Invert and store at 4°C

Miniprep
With QIAprep Spin Miniprep Kit (QIAGEN)

Materials

• Bacterial overnight liquid cultures (1 - 5 mL)
• QIAprep Spin Miniprep Kit

Procedure

1. Pellet 1 -5 mL bacterial culture by centrifugation at more than 8000 rpm for 3 minutes
2. Resuspend pelleted bacterial cells in 250 μL P1 buffer and transfer to a microcentrifuge tube
3. Add 250 μL P2 buffer and mix by inverting tube 4 – 6 times
4. Add 350 μL N3 buffer and mix by inverting tube 4- 6 times
5. Centrifuge for 10 min at 13000 rpm
6. Apply supernatant to the QIAprep spin column by pipetting, centrifuge for 30 – 60 seconds and discard flow-through
7. Wash the QIAprep spin column by adding 0.5 mL PB buffer, centrifuge for 30 – 60 seconds and discard flow-through
8. Wash the QIAprep spin column by adding 0.75 mL PE buffer, centrifuge for 30 – 60 seconds and discard flow-through
9. Centrifuge for 1 minute to remove residual wash buffer
10. Elute DNA by placing QIAprep column in a clean 1.5 mL microcentrifuge tube and adding 50 μL EB buffer or water (or less for higher concentration). Let stand for 1 minute and centrifuge for 1 minute

Comments

With low copy plasmid it is often difficult to obtain high concentrations (needed for sequencing). In these cases it's possible to proceed as follows: culture bacteria in 5 x 7mL tubes of LB medium and perform the miniprep for each tube separately until step 9. Then elute DNA by applying two times step 10 to each tube with the same 50µl of EB buffer.

Polymerase Chain Reaction (PCR)

Restriction Digest
"Typical" Restriction Digest based on NEB Protocol

Materials

• Restriction Enzyme(s)
• DNA
• 10X NEBuffer (Appropriate buffer for used enzyme)
• Water

Procedure

1. Prepare following reaction in 0.5 mL PCR tubes, adding enzyme(s) last:

Component	20 μL reaction	50 μL reaction
Restriction enzyme(s)	1 μL (for each enzyme)	1 μL (for each enzyme)
DNA	100 ng - 1 μg	100 ng - 1 μg
10X NEBuffer	2 μL	5 μL
Water	to 20 μL	to 50 μL

20 μL reactions are sufficient for restriction enzyme analysis, larger volumes are usefull if product is used for cloning

2. Incubate at temperature and for duration appropriate for used enzyme (typically 37°C for 15 minutes or 1 hour)
3. Optional: Inactivate enzyme by incubating reaction at temperature and for duration appropriate for used enzyme (typically 65°C for 20 minutes)

S. Cerevisiae Specific Protocols

Site-directed mutagenesis
Based on NEB Q5 Site-directed Mutagensis Kit Protocol

Materials

• Q5 Hot Start High-Fidelity 2X Master Mix (NEB)
• Forward and reverse primers
• Template DNA
• Nuclease-free water

Procedure

1. Prepare following reaction in 0.5 mL PCR tubes on ice, adding Mater Mix last:

Component	25 μL reaction
Q5 Hot Start High-Fidelity 2X Master Mix	12.5 μL
10 μM Forward Primer	1.25 μL
10 μM Reverse Primer	1.25 μL
Template DNA (1-25 ng/μL)	1 μL
Nuclease-free Water	9 μL

2. Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:

Step		Temperature	Time
Initial Denaturation		98°C	30 seconds
25 cycles	Denaturation	98°C	10 seconds
	Annealing	50 – 72°C	10 – 30 seconds
	Extension	72°C	20 - 30 seconds per kb
Final Extension		72°C	2 minutes
Hold		4°C

3. Assemble following reagents:

Component	Volume
PCR Product	1 μL
2X KLD Reaction Buffer	5 μL
10X KLD Enzyme Mix	1 μL
Nuclease-free Water	3 μL

4. Mix by pipetting up and down and incubate at room temperature for 5 minutes
5. Thaw completent E. coli cells on ice
6. Add 5 μL of KLD mix to the tube of thawed cells. Flick tube 4-5 times to mix.
7. Place mixture on ice for 30 minutes
8. Heat shock at 42°C for 30 seconds
9. Place on ice for 5 minutes
10. Pipette 950 μL of room temperature SOC into the mixture
11. Incubate at 37°C for 60 minutes with shaking
12. Mix thouroughly by flicking the tube and inverting, then spread 50-100 μL on a plate with appropriate antibiotics and incubate overnight at 37°C

Rapid ligation using T4 ligase

Materials

• 5X ligase reaction buffer
• vector DNA
• insert DNA
• autoclaved distilled water
• T4 DNA ligase

Procedure

1. In a final volume of 20 µL mix 4 µL 5X ligase reaction buffer, 3 to 30 fmol vector DNA, 9 to 90 fmol insert DNA, 1 µL T4 ligase and complete with water
2. Mix gently. Centrifuge briefly to bring the contents to the bottom of the tube
3. Incubate at room temperature for 5 min
4. Use 2 μl of the ligation reaction to transform competent cells

Tris-Acetate-EDTA (TAE) buffer 50X

Materials

• Acetate 100%
• Tris base
• EDTA 0,5M

Procedure (1L)

1. Add 100mL of 0,5M EDTA (pH 8.0)
2. Add 242g of Tris base
3. Add 57,1mL of glacial acetic acid (100%)
4. Fill up to 1L with water (adjust pH to ~8.3)
5. Send to autoclave

To prepare 1L of 1X TAE dilute 20mL of 50X TAE in 980mL of water.

Transformation
Based on NEB Transformation Protocol

Materials

• Competent cells
• DNA
• SOC medium (SOB + Glucose)
• Petri dish with appropriate antibiotic resistance

Procedure

1. Thaw competent cells on ice
2. Add 2 μL DNA to the competent cells, mix by pipetting up and down or flicking the tube 4-5 times
3. Place mixture on ice for 30 minutes
4. Heat shock at 42°C for 30 seconds
5. Transfer tubes to ice for 2 minutes
6. Add 950 μL room-temperature SOC media
7. Incubate at 37°C for 60 minutes with shaking
8. Spread 100 μL cells onto selection plates (warm plates to 37°C prior to this step for increased efficiency)
9. Incubate overnight at 37°C