Team:EPF Lausanne/Notebook/Protocols

Protocols

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

Amino acid solution

Materials

• Histidine-Hcl
• Uracil
• Leucine
• Tryptophan

Procedure

Stock concentration	Final concentration	Total quantity for 50 mL
100 mM Histidine-Hcl (209 g/mol)	20.9 g/L	1.045 g
20 mM Uracil (112 g/mol)	2.24 g/L	0.112 g
100 mM Leucine (131 g/mol)	13.1 g/L	0.655 g
40 mM Tryptophan (204 g/mol)	8.16 g/L	0.408 g

1. Filter and sterilize solutions
2. Add 8 mL per liter of selective medium or spread 500 μL on a selective plate

Colony PCR

Materials

• Materials for Taq PCR (except template plasmid DNA)
• Petri dish with transformed colonies

Procedure

1. Prepare 25 μL reactions as in Taq PCR Protocol without template DNA
2. With a sterile tip, under the flame, scrape part of a single colony and add to PCR tubes
3. Mix by pipetting up and down or flicking the reactions
4. Put tubes in thermocycler with cycling conditions as described in Taq PCR Protocol with a longer initial denaturation (2 - 5 minutes)

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

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.

Phusion PCR
Based on NEB Phusion PCR Protocol

Materials

• 5X Phusion HF or GC Buffer
• dNTPs
• Forward and Reverse Primers
• Template plasmid DNA
• Phusion DNA polymerase
• Nuclease Free Water

Procedure

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

Component	20 μL reaction	50 μL reaction
5X Phusion HF or GC Buffer	4 μL	10 μL
10 mM dNTPs	0.4 μL	1 μL
10 μM Forward Primer	1 μL	2.5 μL
10 μM Reverse Primer	1 μL	2.5 μL
Template plasmid DNA	1 pg – 10 ng	1 pg – 10 ng
Phusion DNA Polymerase	0.2 μL	0.5 μL
Nuclease Free Water	to 20 μL	to 50 μL

Usually 100 pg – 1 ng of template DNA is sufficient

2. Mix by pipetting up and down or flicking the reactions
3. Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:

Step		Temperature	Time
Initial Denaturation		98°C	30 seconds
25 – 35 cycles	Denaturation	98°C	5 - 10 seconds
	Annealing	45 – 72°C	10 – 30 seconds
	Extension	72°C	15 -30 seconds per kb
Final Extension		72°C	5 -10 minutes
Hold		4°C

Guidelines

To be completed

Taq PCR
Based on NEB Taq PCR Protocol

Materials

• 10X Standard Taq Reaction Buffer
• dNTPs
• Forward and Reverse Primers
• Template plasmid DNA
• Taq DNA polymerase
• Nuclease Free Water

Procedure

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

Component	25 μL reaction	50 μL reaction
10X Standard Taq Reaction Buffer	2.5 μL	5 μL
10 mM dNTPs	0.5 μL	1 μL
10 μM Forward Primer	0.5 μL	1 μL
10 μM Reverse Primer	0.5 μL	1 μL
Template plasmid DNA	1 pg – 1 ng	1 pg – 1 ng
Taq DNA Polymerase	0.125 μL	0.25 μL
Nuclease Free Water	to 25 μL	to 50 μL

Usually 100 pg – 1 ng of template DNA is sufficient

2. Mix by pipetting up and down or flicking the reactions
3. Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:

Step		Temperature	Time
Initial Denaturation		95°C	30 seconds
25 – 35 cycles	Denaturation	95°C	15 – 30 seconds
	Annealing	45 – 68°C	15 – 60 seconds
	Extension	68°C	1 minutes per kb
Final Extension		68°C	5 minutes
Hold		4°C

Guidelines

To be completed

Q5 PCR
Based on NEB Q5 PCR Protocol

Materials

• 5X Q5 Reaction Buffer
• dNTPs 10µM
• Forward and Reverse Primers
• Template plasmid DNA
• Q5 High Fidelity DNA polymerase
• Nuclease Free Water

Procedure

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

Component	25 μL reaction	50 μL reaction
5X Q5 Reaction Buffer	5 μL	10 μL
10 mM dNTPs	0.5 μL	1 μL
10 μM Forward Primer	1.25 μL	2.5 μL
10 μM Reverse Primer	1.25 μL	2.5 μL
Template plasmid DNA	1 < 1,000 ng	1 < 1,000 ng
Q5 DNA Polymerase	0.25 μL	0.5 μL
Nuclease Free Water	to 25 μL	to 50 μL

Usually 100 pg – 1 ng of template DNA is sufficient

2. Mix by pipetting up and down or flicking the reactions
3. Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:

Step		Temperature	Time
Initial Denaturation		98°C	30 seconds
25 – 35 cycles	Denaturation	98°C	5 – 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

Guidelines

To be completed

PCR Product Purification
With QIAquick PCR Purification Kit (QIAGEN)

Materials

• PCR products
• QIAquick PCR Purification Kit

Procedure

1. Add 5 volumes PB buffer to 1 volume of PCR product and mix
2. Place QIAquick column in 2 ml collection tube
3. Apply samples to QIAquick column and centrifuge for 30 – 60 seconds, discard flow-through
4. Wash by adding 750 μL PE buffer to QIAquick column and centrifuge fo 30 – 60 seconds, discard flow-through
5. Centrifuge QIAquick column for 1 minutes to remove residual wash buffer
6. Elute DNA by adding 30 or 50 μL EB buffer or water to the center of the QIAquick column. Let stand for 1 minutes and centrifuge for 1 minute

PEG/LiAc Solution

Materials

• 50% PEG (Polyethylene glycol) prepared with sterile deionized water
• 10X TE buffer: 0.1 M Tris-Hcl, 10 mM EDTA, ph 7.5, autoclaved
• 10X LiAc: 1 M lithium acetate, pH 7.5 adjusted with dilute acetic acid, autoclaved

Procedure

1. Prepare PEG/LiAc solution as follows:

Stock concentration	Final concentration	Total quantity for 10 mL solution
50% PEG	40% PEG	8 mL
10X TE buffer	1X TE buffer	1 mL
10X LiAc	1X LiAc	1 mL

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)

Sd Medium

Materials

• Amino Acid Powder
• Yeast Nitrogen Base
• Ammonium Sulphate
• Adenine Sulphate
• Water
• NaOH
• Agar
• Glucose

Procedure

1. Place stirrer bar in 2 L Erlenmeyer
2. Add 2.6 g amino acid powder, 3.4 g yeast nitrogen base, 10 g ammonium sulphate, 1 g adenine sulphate and 950 mL water
3. Adjust pH to 5.9 by adding a few drops of 10 M NaOH
4. In an other Erlenmeyer, add 35 g agar and 900 mL water
5. Autoclave both bottles
6. Transfer the content of first bottle to the agar-containing bottle
7. Cool to 55°C
8. Add 100 ml 40% glucose and 16 ml of the required amino acids
9. Pour plates

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