Latest revision as of 23:45, 16 September 2015

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

Protocols

Agarose Gel
Competent Cell Preparation
5' Dephosphorylation
Gel Extraction
Gibson Assembly
Glycerol Stock
Lysogeny Broth (LB) Medium
Lysogeny Broth (LB) Agar Plates
Miniprep
Polymerase Chain Reaction (PCR)
Restriction Digest
S. cerevisiae Specific Protocols
Site-directed mutagenesis
T4 ligation
Tris-Acetate-EDTA (TAE) buffer 50X
Transformation

Agarose Gel

Materials

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

Procedure

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

Competent Cell Preparation
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

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

5' Dephosphorylation
NEB Protocol

Materials

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

Procedure

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

Gel extraction
QIAGEN protocol

Materials

Agarose gel
QIAquick Gel Extraction Kit

Procedure

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
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
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
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
Add 1 gel volume of isopropanol to the sample and mix
Place a QIAquick spin column in a provided 2 mL collection tube
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
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
Centrifuge the QIAquick column once more in the provided 2 ML collection tube for 1 min at 13'000 rpm to remove residual buffer
Place QIAquick column into a clean 1.5 mL microcentrifuge tube
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
NEB Protocol

Materials

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

Procedure

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

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

Glycerol Stock
Addgene Protocol

Materials

50% Glycerol
Bacterial overnight liquid culture
Liquid Nitrogen

Procedure

Mix 0.5 mL 50% glycerol and 0.5 mL bacterial culture
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)

Dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl in 950 mL deionized water
Adjust pH to 7 using 1 M NaOH and bring volume to 1 L
Autoclave
If necessary: let medium cool to 55°C and add antibiotic
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)

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

Miniprep
QIAGEN Protocol

Materials

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

Procedure

Pellet 1 -5 mL bacterial culture by centrifugation at more than 8000 rpm for 3 minutes
Resuspend pelleted bacterial cells in 250 μL P1 buffer and transfer to a microcentrifuge tube
Add 250 μL P2 buffer and mix by inverting tube 4 – 6 times
Add 350 μL N3 buffer and mix by inverting tube 4- 6 times
Centrifuge for 10 min at 13000 rpm
Apply supernatant to the QIAprep spin column by pipetting, centrifuge for 30 – 60 seconds and discard flow-through
Wash the QIAprep spin column by adding 0.5 mL PB buffer, centrifuge for 30 – 60 seconds and discard flow-through
Wash the QIAprep spin column by adding 0.75 mL PE buffer, centrifuge for 30 – 60 seconds and discard flow-through
Centrifuge for 1 minute to remove residual wash buffer
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)

Colony PCR

Materials

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

Procedure

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

Phusion PCR
NEB Protocol

Materials

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

Procedure

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

Mix by pipetting up and down or flicking the reactions
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

Taq PCR
NEB Protocol

Materials

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

Procedure

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

Mix by pipetting up and down or flicking the reactions
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

Q5 PCR
NEB Protocol

Materials

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

Procedure

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

Mix by pipetting up and down or flicking the reactions
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

PCR Product Purification
QIAGEN Protocol

Materials

PCR products
QIAquick PCR Purification Kit

Procedure

Add 5 volumes PB buffer to 1 volume of PCR product and mix
Place QIAquick column in 2 ml collection tube
Apply samples to QIAquick column and centrifuge for 30 – 60 seconds, discard flow-through
Wash by adding 750 μL PE buffer to QIAquick column and centrifuge fo 30 – 60 seconds, discard flow-through
Centrifuge QIAquick column for 1 minutes to remove residual wash buffer
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

Restriction Digest
NEB Protocol

Materials

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

Procedure

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

Incubate at temperature and for duration appropriate for used enzyme (typically 37°C for 15 minutes or 1 hour)
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

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

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

Yeast integration

Procedure

Start a pre-culture of 50mL YPD or SD + Glucose with one colony. Growing at 30°C with shaking (250 rpm) for 16h to 18h untill stationary phase, OD600 > 1,5.
Seed yeast in 500 mL YPD or SD + Glucose. OD should be 0,2. Grow at 30°C in shaker for 3 hours untill OD 0,4 - 0,6.
Spin yeast down 5 min at 800 x g (1800 rpm approx).
Pour off medium and check that the medium is clear (if not, you may have a bacterial contamination).
Wash yeast with 25 mL of water (dH2O). Resuspend and pool cells into one tube.
Spin yeast down, 5 min at 1800 rpm. Decant supernatant.
Resuspend yeasts in freshly prepared TE/LiAc solution (approx 25 mL)
Spin yeast down, 5 min at 1800 rpm. Decant supernatant.
Resuspend yeast in TE/LiAc solution according to the following formula: OD600 x culture volume/60 = vol in which to resuspend (around 2 mL)
Add 10% vol of ssDNA (boiled at 100°C for 5 min and cooled on ice).
Add linearized vectors to the yeast (1 μg).
Add 1 ml of TE/lithium acetate/PEG solution, and resuspend carefully.
Incubate 30 min at 30°C with shaking 200 rpm.
Heat-shock the cells for exactly 20’ at 42°C (in a water bath).
Centrifuge the tubes for 5’’ in a microfuge at full speed at room temperature; remove the supernatant
Resuspend the cells in 50µl of sterile water, and plate onto the appropriate selective media plates using glass beads. Need 2-3 days to grow, at 30°C.

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

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

Sd Medium

Materials

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

Procedure

Place stirrer bar in 2 L Erlenmeyer
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
Adjust pH to 5.9 by adding a few drops of 10 M NaOH
In an other Erlenmeyer, add 35 g agar and 900 mL water
Autoclave both bottles
Transfer the content of first bottle to the agar-containing bottle
Cool to 55°C
Add 100 ml 40% glucose and 16 ml of the required amino acids
Pour plates

ypd medium
Clontech Protocol

Procedure

Prepare YPD mixture as follows :

20 g/L peptone
10 g/L Yeast extract
20 g/L Agar (for plates only)
[Optional] For adenine-supplemented YPD (YPDA), add 15 ml of a 0.2% adenine hemisulfate solution per liter of medium (final concentration is 0.003%, in addition to the trace amount of Ade that is naturally present in YPD). Adenine hemisulfate tolerates autoclaving.
Add H2O to 950 ml. Adjust the pH to 6.5 if necessary, then autoclave. Allow medium to cool to ~ 55°C and then add dextrose (glucose) to 2% (50 ml of a sterile 40% stock solution). Adjust the final volume to 1 L if necessary.
[Optional] For kanamycin-containing medium, prepare YPD or YPDA as above. After autoclaved medium has cooled to 55°C, add 0.2–0.3 ml of 50 mg/ml kanamycin (final concentration 10–15 mg/L)

Site-directed mutagenesis
NEB Protocol

Materials

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

Procedure

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

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

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

Mix by pipetting up and down and incubate at room temperature for 5 minutes
Thaw completent E. coli cells on ice
Add 5 μL of KLD mix to the tube of thawed cells. Flick tube 4-5 times to mix.
Place mixture on ice for 30 minutes
Heat shock at 42°C for 30 seconds
Place on ice for 5 minutes
Pipette 950 μL of room temperature SOC into the mixture
Incubate at 37°C for 60 minutes with shaking
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

Based on Invitrogen protocol (for cohesive ends)

Materials

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

Procedure

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
Mix gently. Centrifuge briefly to bring the contents to the bottom of the tube
Incubate at room temperature for 5 min
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)

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

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

Transformation
NEB Protocol

Materials

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

Procedure

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

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

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+     <div class="row">
+        <div class="col-md-2 scrollspy">
+            <ul id="nav" class="nav hidden-xs hidden-sm" data-spy="affix">
+                <li class="active"><a href="#agarosegel">Agarose Gel</a></li>
+                <li><a href="#competentcells">Competent Cell Preparation</a></li>
+                <li><a href="#dephosphorylation">5' Dephosphorylation</a></li>
+                <li><a href="#gelextraction">Gel Extraction</a></li>
+                <li><a href="#gibsonassembly">Gibson Assembly</a></li>
+                <li><a href="#glycerolstock">Glycerol Stock</a></li>
+                <li><a href="#lbmedium">Lysogeny Broth (LB) Medium</a></li>
+                <li><a href="#lbagar">Lysogeny Broth (LB) Agar Plates</a></li>
+                <li><a href="#miniprep">Miniprep</a></li>
+                <li><a href="#pcr">Polymerase Chain Reaction (PCR)</a>
+                    <ul class="nav">
+                        <li><a href="#colonypcr">Colony PCR</a></li>
+                        <li><a href="#phusionpcr">Phusion PCR</a></li>
+                        <li><a href="#taqpcr">Taq PCR</a></li>
+                        <li><a href="#Q5pcr">Q5 PCR</a></li>
+                        <li><a href="#pcrpurification">PCR Product Purification</a></li>
+                    </ul>
+                </li>
+                <li><a href="#restriction">Restriction Digest</a></li>
+                <li><a href="#yeast">S. cerevisiae Specific Protocols</a>
+                    <ul class="nav">
+                        <li><a href="#aminoacidsolution">Amino Acid Solution</a></li>
+                        <li><a href="#integration">Integration</a></li>
+                        <li><a href="#pegliac">PEG/LiAc Solution</a></li>
+                        <li><a href="#sdmedium">SD Medium</a></li>
+                        <li><a href="#ypdmedium">YPD Medium</a></li>
+                    </ul>
+                </li>
+                <li><a href="#mutagenesis">Site-directed mutagenesis</a></li>
+                <li><a href="#t4ligation">T4 ligation</a></li>
+                <li><a href="#tae">Tris-Acetate-EDTA (TAE) buffer 50X</a></li>
+                <li><a href="#transformation">Transformation</a></li>
+            </ul>
+        </div>
+        <div class="col-md-offset-1 col-md-9">
-    <!-- Footer -->
+<!-- AGAROSE GEL -->
-    <link rel="stylesheet" href="https://2015.igem.org/Team:EPF_Lausanne/css/footer?action=raw&amp;ctype=text/css" type="text/css">
+                <section id="agarosegel" class="panel">
+                <h1>Agarose Gel</h1>
+                <h2>Materials</h2>
+                <ul>
+                    <li>1X TAE</li>
+                    <li>Agarose</li>
+                    <li>Gel Red</li>
+                    <li>DNA samples</li>
+                    <li>6X loading dye</li>
+                    <li>Nuclease-free water</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Prepare 1.2% agarose gel for small fragments and 3% agarose gel for large fragments</li>
+                    <li>Mix 50 mL 1X TAE and 0.6 g (1.2%) or 1.5 g (3%) agarose</li>
+                    <li>Melt in microwave until agarose has melted (about 50 seconds)</li>
+                    <li>Add 1.3 μL (1.2%) or 1.5 μL (3%) Gel Red</li>
+                    <li>Pour solution into agarose gel mold with comb</li>
+                    <li>Let set for 20 minutes or until solid</li>
+                    <li>Place gel in 1X TAE and remove comb</li>
+                    <li>Load samples of 200 ng (or 2  μL) DNA mixed with 2  μL 6X loading dye and nuclease-free water up to 12  μL</li>
+                    <li>Run gel at 100-120 Volts for 40-50 minutes (1.2%) or 80 Volts for 2 hours (3%)</li>
+                    <li>Take a picture of the gel at the UV detector</li>
+                </ol>
+            </section>
-    <script src="https://2015.igem.org/Team:EPF_Lausanne/Test/js/mordernizr-2.6.2?action=raw&amp;ctype=text/js"></script>
-</head>
+<!-- COMPETENT CELL -->
-<body  class="loading">
+            <section id="competentcells" class="panel">
-    <!--[if lt IE 7]>
+                <a href="http://openwetware.org/wiki/Gill:Calcium_chloride_competent_cells"><h1>Competent Cell Preparation</br><small><u>Open Wet Ware Protocol</u></small></h1></a>
-    <p class="chromeframe">You are using an <strong>outdated</strong> browser. Please <a href="http://browsehappy.com/">upgrade your browser</a> or <a href="http://www.google.com/chromeframe/?redirect=true">activate Google Chrome Frame</a> to improve your experience.</p>
+                <h2>Materials</h2>
-    <![endif]-->
+                <ul>
+                    <li>Bacterial overnight liquid culture</li>
+                    <li>Lysogeny broth (LB) medium</li>
+                    <li>CaCl2 solution, ice cold: 60 mM CaCl2, 15% glycerol, 10 mM PIPES, pH 7, filter sterilize and store at room temperature</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Subculture overnight culture 1:100 in LB medium</li>
+                    <li>Incubate at 37°C with shaking until culture reaches an OD600 of 0.375</li>
+                    <li>Aliquot 20 mL if the culture into chilled 50 mL tubes</li>
+                    <li>Leave tubes on ice for 5 – 10 minutes</li>
+                    <li>Centrifuge cells at 1600 g for 7 minutes at 4°C</li>
+                    <li>Discard supernatant and resuspend pellet in 4 mL ice cold CaCl2 solution</li>
+                    <li>Centrifuge cells at 1100 g for 5 minutes at 4°C</li>
+                    <li>Discard supernatant and resuspend pellet in 4 mL ice cold CaCl2 solution</li>
+                    <li>Keep on ice for 30 minutes</li>
+                    <li>Centrifuge cells at 1100 g for 5 minutes at 4°C</li>
+                    <li>Discard supernatant and resuspend pellet in 800 μL ice cold CaCl2 solution</li>
+                    <li>Aliquot 100 μL of this suspension into microcentrifuge tubes</li>
+                    <li>Freeze in liquid nitrogen and store at -80°C</li>
+                </ol>
+            </section>
-    <!-- HEADER -->
+<!-- DEPHOSPHORYLATION -->
-    <div class="site-header">
+                         <section id="dephosphorylation" class="panel">
-        <div class="container">
+                             <a href="https://www.neb.com/protocols/1/01/01/vector-dephosphorylation-protocol"><h1>5' Dephosphorylation</br><small><u>NEB Protocol</u></small></h1></a>
-            <div class="row">
+                             <h2>Materials</h2>
-                <nav class="navbar navbar-default navbar-static-top">
+                            <ul>
-                    <div class="navbar-header">
+                                 <li>1-5 µg cut DNA </li>
-                         <button class="navbar-toggle" type="button" data-toggle="collapse" data-target="#main-menu">
+                                 <li>10X Antarctic Phosphatase Reaction Buffer</li>
-                            <span class="sr-only">Toggle navigation</span>
+                                 <li>Antarctic Phosphatase</li>
-                             <i class="fa fa-bars"></i>
-                        </button>
-                        <a class="navbar-brand" href="https://2015.igem.org/Team:EPF_Lausanne">Bio Logic</a>
-                    </div>
-                    <div class="collapse navbar-collapse" id="main-menu">
-                        <ul class="nav navbar-nav navbar-right">
-                            <li><a href="https://2015.igem.org/Team:EPF_Lausanne">Home</a></li>
-                            <li class="dropdown">
-                            <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-haspopup="true" aria-expanded="false">Project <span class="caret"></span></a>
-                             <ul class="dropdown-menu">
-                                <li><a href="https://2015.igem.org/Team_EPF_Lausanne/Project/Background">Background</a></li>
-                                 <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Project/Description">Description</a></li>
-                                 <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Project/Applications">Applications</a></li>
-                                 <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Project/Modelling">Modelling</a></li>
                              </ul>
-                             </li>
+                             <h2>Procedure</h2>
-                            <li class="dropdown">
+                             <ol>
-                            <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-haspopup="true" aria-expanded="false">Achievements <span class="caret"></span></a>
+                                 <li>Add 1/10 volume of 10X Antarctic Phosphatase Reaction Buffer to 1-5 µg of DNA cut with any restriction endonuclease in any buffer.</li>
-                             <ul class="dropdown-menu">
+                                 <li>Add 1 µl of Antarctic Phosphatase (5 units) and mix</li>
-                                 <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Achievements/Vivo">In vivo</a></li>
+                                 <li>Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions</li>
-                                 <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Achievements/Silico">In silico</a></li>
+                                 <li>Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 70°C</li>
-                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Achievements/Judging">Judging</a></li>
+                                 <li>Proceed with ligation</li>
-                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Achievements/Parts">Parts</a></li>
+                             </ol>
-                            </ul>
+                         </section>
-                            </li>
-                            <li class="dropdown">
-                            <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-haspopup="true" aria-expanded="false">Policy and Practices <span class="caret"></span></a>
-                            <ul class="dropdown-menu">
-                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Policy/Ethic">Ethics</a></li>
-                                 <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Policy/Safety">Safety</a></li>
-                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Policy/Human">Human Practices</a></li>
-                            </ul>
-                            </li>
-                            <li class="dropdown">
-                            <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-haspopup="true" aria-expanded="false">Notebook <span class="caret"></span></a>
-                            <ul class="dropdown-menu">
-                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Notebook/Ecoli">E. Coli</a></li>
-                                 <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Notebook/Yeast">Yeast</a></li>
-                                 <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Notebook/Protocols">Protocols</a></li>
-                             </ul>
-                            </li>
-                            <li class="dropdown">
-                            <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-haspopup="true" aria-expanded="false">Team <span class="caret"></span></a>
-                            <ul class="dropdown-menu">
-                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Team/Meet">Meet us</a></li>
-                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Timeline">Timeline</a></li>
-                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Team/Aknowledgements">Aknowledgements</a></li>
-                            </ul>
-                            </li>
-                         </ul>
-                    </div>
-                </nav>
-            </div>
-        </div>
-    </div> <!-- .site-header -->
-    <div class="page-h promotion">
-        <div class="container">
-            <div class="row">
-                <div class="col-md-12 text-center">
-                    <h3>Protocols</h3>
-                </div>
-            </div>
-        </div>
-    </div>
-    <div class="first-section">
-        <div class="row">
-             <div class="col col-md-3">
-                <nav id="affix-nav" class="sidebar hidden-sm hidden-xs">
-                    <ul class="nav sidenav box" data-spy="affix" data-offset-top="200" data-offset-bottom="600">
-                        <li class="active"><a href="#gel_protocol">Agarose Gel Preparation</a></li>
-                        <li><a href="#amino_acid_solution">Amino acid solutions Protocol</a></li>
-                        <li><a href="#colony_pcr">Colony PCR</a></li>
-                        <li><a href="#gibson_protocol">Gibson Assembly Protocol</a></li>
-                        <li><a href="#miniprep_protocol">Miniprep – with QlAprep Spin Miniprep kit</a></li>
-                        <li><a href="#pcr_protocol">PCR Protocol</a>
-                        <ul class="nav">
-                          <li><a href="#taq">Taq PCR</a></li>
-                        </ul>
-                        </li>
-                        <li><a href="#purification_protocol">PCR Product Purification Protocol</a></li>
-                        <li><a href="#PEG_LiAc_solution">PEG/LiAc solution protocol</a></li>
-                        <li><a href="#sdmedium_protocol">SD Medium</a></li>
-                        <li><a href="#competent_protocol">Thermocompetent <i>E. coli</i> cells preparation</a>
-                        <ul class="nav">
-                          <li><a href="#inouebuffer">Inoue Transformation Buffer</a></li>
-                        </ul>
-                        </li>
-                        <li><a href="#transformation_protocol">Transformation Protocols</a>
-                       <ul class="nav">
-                          <li><a href="#NEB">NEB transformation protocol</a></li>
-                          <li><a href="#igem">iGEM Transformation Protocol</a></li>
-                        </ul>
-                        </li>
-                        <li><a href="#yeast_transformation_protocol">Yeast transformation and homolgous recombination procedure from Clontech Yeast Handbook</a></li>
-                        <li><a href="#YPD_medium_protocol">YPD Medium protocol</a></li>
-                    </ul>
-                </nav>
-            </div>
-        </div>
-    </div>
-            <!--<div class="col-md-3" id="leftCol">
-                <div class="whitebg box">
-                    <ul class="nav sidebar hidden-sm hidden-xs" id="sidebar">
+<!-- GEL EXTRACTION -->
-                        <li>
+                        <section id="gelextraction" class="panel">
-                             <a href="#Prot1">Protocol 1</a>
+                             <a href="https://www.qiagen.com/ch/resources/resourcedetail?id=f4ba2d24-8218-452c-ad6f-1b6f43194425&lang=en"><h1>Gel extraction</br><small><u>QIAGEN protocol</u></small></h1></a>
-                            <ul class="nav">
+                            <h2>Materials</h2>
-                              <li class=""><a href="#">- Sub 1</a></li>
+                            <ul>
-                              <li class=""><a href="#">- Sub 2</a></li>
+                                <li>Agarose gel </li>
+                                <li>QIAquick Gel Extraction Kit </li>
                              </ul>
-                        </li>
-                        <li><a href="#Prot2">Protocol 2</a></li>
-                        <li><a href="#Prot3">Protocol 3</a></li>
-                        <li><a href="#Prot4">Protocol 4</a></li>
-                        <li><a href="#Prot5">Protocol 5</a></li>
-                        <li><a href="#Prot6">Protocol 6</a></li>
-                        <li><a href="#Prot7">Protocol 7</a></li>
-                        <li><a href="#Prot8">Protocol 8</a></li>
-                        <li><a href="#Prot9">Protocol 9</a></li>
-                        <li><a href="#Prot10">Protocol 10</a></li>
-                    </ul>
-                </div>
-            </div>
--->
-            <div class="col-md-8">
-                <div id="Prot1" class="whitebg box">
-                    <h3>Protocol #1</h3>
-                    <p>Materials</p>
-                    <ul>
-                        <li>First item</li>
-                        <li>Second one</li>
-                    </ul>
-                    <p>Procedure</p>
-                    <ol>
-                        <li>Take the first item</li>
-                        <li>Put it in the second</li>
-                        <li>Voilà</li>
-                    </ol>
-                </div>
-                <div id="Prot2" class="whitebg box">
+                            <h2>Procedure</h2>
-                    <h3>Protocol #2</h3>
+                            <ol>
-                    <p>Materials</p>
+                                <li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel</li>
-                    <ul>
+                                <li>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</li>
-                        <li>First item</li>
+                                <li> 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</li>
-                        <li>Second one</li>
+                                <li>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</li>
-                    </ul>
+                                <li>Add 1 gel volume of isopropanol to the sample and mix</li>
-                    <p>Procedure</p>
+                                <li>Place a QIAquick spin column in a provided 2 mL collection tube</li>
-                    <ol>
+                                <li>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</li>
-                        <li>Take the first item</li>
+                                <li>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</li>
-                        <li>Put it in the second</li>
+                                <li>Centrifuge the QIAquick column once more in the provided 2 ML collection tube for 1 min at 13'000 rpm to remove residual buffer</li>
-                        <li>Voilà</li>
+                                <li> Place QIAquick column into a clean 1.5 mL microcentrifuge tube</li>
-                    </ol>
+                                <li> 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.</li>
-                </div>
+                            </ol>
-                <div id="Prot3" class="whitebg box">
+                         </section>
-                    <h3>Protocol #3</h3>
-                    <p>Materials</p>
-                    <ul>
-                         <li>First item</li>
-                        <li>Second one</li>
-                    </ul>
-                    <p>Procedure</p>
-                    <ol>
-                        <li>Take the first item</li>
-                        <li>Put it in the second</li>
-                        <li>Voilà</li>
-                    </ol>
-                </div>
-                <div id="Prot4" class="whitebg box">
-                    <h3>Protocol #4</h3>
-                    <p>Materials</p>
-                    <ul>
-                        <li>First item</li>
-                        <li>Second one</li>
-                    </ul>
-                    <p>Procedure</p>
-                    <ol>
-                        <li>Take the first item</li>
-                        <li>Put it in the second</li>
-                        <li>Voilà</li>
-                    </ol>
-                </div>
-                <div id="Prot5" class="whitebg box">
-                    <h3>Protocol #5</h3>
-                    <p>Materials</p>
-                    <ul>
-                        <li>First item</li>
-                        <li>Second one</li>
-                    </ul>
-                    <p>Procedure</p>
-                    <ol>
-                        <li>Take the first item</li>
-                        <li>Put it in the second</li>
-                        <li>Voilà</li>
-                    </ol>
-                </div>
-                <div id="Prot6" class="whitebg box">
+<!-- GIBSON ASSEMBLY -->
-                    <h3>Protocol #6</h3>
+            <section id="gibsonassembly" class="panel">
-                    <p>Materials</p>
+                <a href="https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510"><h1>Gibson Assembly</br><small><u>NEB Protocol</u></small></h1></a>
-                    <ul>
+                <h2>Materials</h2>
-                        <li>First item</li>
+                <ul>
-                        <li>Second one</li>
+                    <li>DNA fragments</li>
-                     </ul>
+                    <li>2X Gibson Assembly Mater Mix (NEB)</li>
-                     <p>Procedure</p>
+                     <li>2X NEBuilder Positive Control (NEB)</li>
-                    <ol>
+                     <li>Deionized water</li>
-                        <li>Take the first item</li>
+                </ul>
-                         <li>Put it in the second</li>
+                <h2>Procedure</h2>
-                        <li>Voilà</li>
+                <ol>
-                     </ol>
+                    <li>Set up following reactions on ice, adding Gibson Assembly Master Mix last:</li>
-                 </div>
+                    <table cellspacing='0' style="width:75%">
+                         <thead>
+                        <tr>
+                            <th>Component</th>
+                            <th>2 – 3 Fragments Assembly</th>
+                            <th>4 – 6 Fragments Assembly</th>
+                            <th>Positive Control</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>Total Amount of Fragments</td>
+                            <td>0.02 – 0.5 pmols</td>
+                            <td>0.2 – 1 pmols</td>
+                            <td>10 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>2X Gibson Assembly Master Mix</td>
+                            <td>10 μL</td>
+                            <td>10 μL</td>
+                            <td>10 μL</td>
+                        </tr>
+                        <tr>
+                            <td>Deionized water  </td>
+                            <td>to 20 μL</td>
+                            <td>to 20 μL</td>
+                            <td>0 μL</td>
+                        </tr>
+                        </tbody>
+                    </table>
+                    <p><small>Optimized efficiency for 50 – 100 ng of vectors and 2 – 3 fold of excess inserts</small></p>
+                    <li>Incubate samples at 50°C for 15 minutes (2 – 3 fragments) or for 60 minutes (4 – 6 fragments)</li>
+                    <li>Store samples on ice or at -20°C until transformation</li>
+                     <li>Transform competent cells following the Transformation Protocol</li>
+                 </ol>
+            </section>
-                <div id="Prot7" class="whitebg box">
+<!--GLYCEROL STOCK-->
-                    <h3>Protocol #7</h3>
+           <section id="glycerolstock" class="panel">
-                    <p>Materials</p>
+                <a href="https://www.addgene.org/plasmid-protocols/create-glycerol-stock/"><h1>Glycerol Stock</br><small><u>Addgene Protocol</u></small></h1></a>
-                    <ul>
+                <h2>Materials</h2>
-                        <li>First item</li>
+                <ul>
-                        <li>Second one</li>
+                    <li>50% Glycerol</li>
-                     </ul>
+                    <li>Bacterial overnight liquid culture</li>
-                    <p>Procedure</p>
+                     <li>Liquid Nitrogen</li>
-                    <ol>
+                </ul>
-                        <li>Take the first item</li>
+                <h2>Procedure</h2>
-                        <li>Put it in the second</li>
+                <ol>
-                        <li>Voilà</li>
+                    <li>Mix 0.5 mL 50% glycerol and 0.5 mL bacterial culture</li>
-                    </ol>
+                    <li>Freeze in liquid nitrogen and store at -80°C</li>
-                </div>
+                </ol>
+            </section>
-                <div id="Prot8" class="whitebg box">
+<!-- LB MEDIUM -->
-                     <h3>Protocol #8</h3>
+            <section id="lbmedium" class="panel">
-                     <p>Materials</p>
+                <h1>Lysogeny Broth (LB) Medium</h1>
-                     <ul>
+                <h2>Materials</h2>
-                        <li>First item</li>
+                <ul>
-                        <li>Second one</li>
+                     <li>Tryptone</li>
-                     </ul>
+                     <li>Yeast extract</li>
-                    <p>Procedure</p>
+                     <li>NaCl</li>
-                    <ol>
+                    <li>Deionized water</li>
-                        <li>Take the first item</li>
+                    <li>NaOH</li>
-                        <li>Put it in the second</li>
+                     <li>If necessary: antibiotics</li>
-                        <li>Voilà</li>
+                </ul>
-                     </ol>
+                <h2>Procedure (for 1 L)</h2>
-                 </div>
+                <ol>
+                    <li>Dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl in 950 mL deionized water</li>
+                    <li>Adjust pH to 7 using 1 M NaOH and bring volume to 1 L</li>
+                    <li>Autoclave</li>
+                     <li>If necessary: let medium cool to 55°C and add antibiotic</li>
+                    <li>Store at room temperature</li>
+                 </ol>
+            </section>
-                <div id="Prot9" class="whitebg box">
+<!-- LB AGAR -->
-                     <h3>Protocol #9</h3>
+            <section id="lbagar" class="panel">
-                     <p>Materials</p>
+                <h1>Lysogeny Broth (LB) Agar Plates</h1>
-                     <ul>
+                <h2>Materials</h2>
-                        <li>First item</li>
+                <ul>
-                        <li>Second one</li>
+                     <li>Tryptone</li>
-                     </ul>
+                     <li>Yeast extract</li>
-                     <p>Procedure</p>
+                     <li>NaCl</li>
-                     <ol>
+                    <li>Deionized water</li>
-                        <li>Take the first item</li>
+                    <li>NaOH</li>
-                        <li>Put it in the second</li>
+                     <li>Agar</li>
-                        <li>Voilà</li>
+                     <li>If necessary: antibiotics</li>
-                     </ol>
+                     <li>Petri dishes</li>
-                 </div>
+                </ul>
+                <h2>Procedure (for 1 L, ie. about 50 plates)</h2>
+                <ol>
+                    <li>Dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl in 950 mL deionized water</li>
+                    <li>Adjust pH to 7 using 1 M NaOH and bring volume to 1 L</li>
+                    <li>Add 15 g agar
+                    <li>Autoclave</li>
+                     <li>If necessary: let medium cool to 55°C and add antibiotic</li>
+                    <li>Pour into petri dishes (about 20 mL per dish) and let set</li>
+                    <li>Invert and store at 4°C</li>
+                 </ol>
+            </section>
-                <div id="Prot10" class="whitebg box">
+<!-- MINIPREP -->
-                    <h3>Protocol #10</h3>
+            <section id="miniprep" class="panel">
-                    <p>Materials</p>
+                <a href="https://www.qiagen.com/ch/resources/resourcedetail?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en"><h1>Miniprep</br><u><small>QIAGEN Protocol</small></u></h1></a>
-                    <ul>
+                <h2>Materials</h2>
-                        <li>First item</li>
+                <ul>
-                        <li>Second one</li>
+                    <li>Bacterial overnight liquid cultures (1 - 5 mL) </li>
-                    </ul>
+                    <li>QIAprep Spin Miniprep Kit </li>
-                    <p>Procedure</p>
+                 </ul>
-                    <ol>
-                        <li>Take the first item</li>
-                        <li>Put it in the second</li>
-                        <li>Voilà</li>
-                    </ol>
-                 </div>
-            </div>
-        </div>
-    <div class="fourth-section">
+                <h2>Procedure</h2>
-        <div class="container">
+                <ol>
-            <div class="row">
+                    <li>Pellet 1 -5 mL bacterial culture by centrifugation at more than 8000 rpm for 3 minutes</li>
-                <div class="col-md-12 text-center">
+                     <li>Resuspend pelleted bacterial cells in 250 μL P1 buffer and transfer to a microcentrifuge tube</li>
-                     <h2>Still under construction</h2>
+                    <li> Add 250 μL P2 buffer and mix by inverting tube 4 – 6 times</li>
-                </div>
+                    <li>Add 350 μL N3 buffer and mix by inverting tube 4- 6 times</li>
-            </div>
+                    <li>Centrifuge for 10 min at 13000 rpm</li>
-        </div>
+                    <li>Apply supernatant to the QIAprep spin column by pipetting, centrifuge for 30 – 60 seconds and discard flow-through</li>
-    </div>
+                    <li>Wash the QIAprep spin column by adding 0.5 mL PB buffer, centrifuge for 30 – 60 seconds and discard flow-through</li>
-    <!-- Footer -->
+                    <li>Wash the QIAprep spin column by adding 0.75 mL PE buffer, centrifuge for 30 – 60 seconds and discard flow-through</li>
-    <footer class="footer-distributed">
+                    <li>Centrifuge for 1 minute to remove residual wash buffer</li>
+                    <li> 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</li>
+                </ol>
-             <div class="footer-left">
+                <h2>Comments</h2>
+                <p>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.</p>
+             </section>
-                <img class="resize50" src="https://static.igem.org/mediawiki/2015/5/59/Logo_iGEM_w.png" alt="">
+<!--PCR-->
+        <section id="pcr" class="panel">
+            <h1>Polymerase Chain Reaction (PCR)</h1>
-             </div>
+<!-- COLONY PCR -->
+            <section id="colonypcr" class="panel">
+                <h1>Colony PCR</h1>
+                <h2>Materials</h2>
+                <ul>
+                    <li>Materials for Taq PCR (except template plasmid DNA)</li>
+                    <li>Petri dish with transformed colonies</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Prepare 25 μL reactions as in Taq PCR Protocol without template DNA</li>
+                    <li>With a sterile tip, under the flame, scrape part of a single colony and add to PCR tubes</lili>
+                    <li>Mix by pipetting up and down or flicking the reactions </li>
+                    <li>Put tubes in thermocycler with cycling conditions as described in Taq PCR Protocol with a longer initial denaturation (2 - 5 minutes)</li>
+                </ol>
+             </section>
-             <div class="footer-center">
+<!-- PHUSION PCR -->
+             <section id="phusionpcr" class="panel">
+                <a href="https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530"><h1>Phusion PCR</br><small><u>NEB Protocol</u></small></h1></a>
+                <h2>Materials</h2>
+                <ul>
+                    <li>5X Phusion HF or GC Buffer</li>
+                    <li>dNTPs</li>
+                    <li>Forward and Reverse Primers</li>
+                    <li>Template plasmid DNA</li>
+                    <li>Phusion DNA polymerase</li>
+                    <li>Nuclease-free Water</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Component</th>
+                            <th>20 μL reaction</th>
+                            <th>50  μL reaction</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>5X Phusion HF or GC Buffer</td>
+                            <td>4 μL</td>
+                            <td>10 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>10 mM dNTPs</td>
+                            <td>0.4 μL</td>
+                            <td>1 μL</td>
+                        </tr>
+                        <tr>
+                            <td>10 μM Forward Primer</td>
+                            <td>1 μL</td>
+                            <td>2.5 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>10 μM Reverse Primer</td>
+                            <td>1 μL</td>
+                            <td>2.5 μL</td>
+                        </tr>
+                        <tr>
+                            <td>Template plasmid DNA</td>
+                            <td>1 pg – 10 ng</td>
+                            <td>1 pg – 10 ng</td>
+                        </tr>
+                        <tr class="even">
+                            <td>Phusion DNA Polymerase</td>
+                            <td>0.2 μL</td>
+                            <td>0.5 μL</td>
+                        </tr>
+                        <tr>
+                            <td>Nuclease-free Water</td>
+                            <td>to 20 μL</td>
+                            <td>to 50 μL</td>
+                        </tr>
+                        </tbody>
+                    </table>
+                    <p><small>Usually 100 pg – 1 ng of template DNA is sufficient</small></p>
+                    <li>Mix by pipetting up and down or flicking the reactions
+                    <li>Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:</li>
+                    <table cellspacing='0' style="width:75%">
+                    <thead>
+                    <tr>
+                        <th>Step</th>
+                        <th>  </th>
+                        <th>Temperature</th>
+                        <th>Time</th>
+                    </tr>
+                    </thead>
+                    <tbody>
+                    <tr>
+                        <td>Initial Denaturation</td>
+                        <td>   </td>
+                        <td>98°C</td>
+                        <td>30 seconds</td>
+                    </tr>
+                    <tr class="even">
+                        <td>25 – 35 cycles</td>
+                        <td>Denaturation</td>
+                        <td>98°C</td>
+                        <td>5 - 10 seconds</td>
+                    </tr>
+                    <tr>
+                        <td>  </td>
+                        <td>Annealing</td>
+                        <td>45 – 72°C</td>
+                        <td>10 – 30 seconds</td>
+                    </tr>
+                    <tr class="even">
+                        <td>   </td>
+                        <td>Extension</td>
+                        <td>72°C</td>
+                        <td>15 -30 seconds per kb</td>
+                    </tr>
+                    <tr>
+                        <td>Final Extension</td>
+                        <td>   </td>
+                        <td>72°C</td>
+                        <td>5 -10 minutes</td>
+                    </tr>
+                    <tr class="even">
+                        <td>Hold</td>
+                        <td>   </td>
+                        <td>4°C </td>
+                        <td>  </td>
+                    </tr>
+                    </tbody>
+                    </table>
+                </ol>
+            </section>
-                <div>
+<!-- TAQ PCR -->
-                    <i class="fa fa-map-marker"></i>
+            <section id="taqpcr" class="panel">
-                     <p><span>Route Cantonale</span> Lausanne, Suisse</p>
+                <a href="https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273"><h1>Taq PCR</br><small><u>NEB Protocol</u></small></h1></a>
-                 </div>
+                <h2>Materials</h2>
+                <ul>
+                     <li>10X Standard Taq Reaction Buffer</li>
+                    <li>dNTPs</li>
+                    <li>Forward and Reverse Primers</li>
+                    <li>Template plasmid DNA</li>
+                    <li>Taq DNA polymerase</li>
+                    <li>Nuclease-free Water</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Component</th>
+                            <th>25 μL reaction</th>
+                            <th>50 μL reaction</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>10X Standard Taq Reaction Buffer</td>
+                            <td>2.5 μL</td>
+                            <td>5 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>10 mM dNTPs</td>
+                            <td>0.5 μL</td>
+                            <td>1 μL</td>
+                        </tr>
+                        <tr>
+                            <td>10 μM Forward Primer</td>
+                            <td>0.5 μL</td>
+                            <td>1 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>10 μM Reverse Primer</td>
+                            <td>0.5 μL</td>
+                            <td>1 μL</td>
+                        </tr>
+                        <tr>
+                            <td>Template plasmid DNA</td>
+                            <td>1 pg – 1 ng</td>
+                            <td>1 pg – 1 ng</td>
+                        </tr>
+                        <tr class="even">
+                            <td>Taq DNA Polymerase</td>
+                            <td>0.125 μL</td>
+                            <td>0.25 μL</td>
+                        </tr>
+                        <tr>
+                            <td>Nuclease-free Water</td>
+                            <td>to 25 μL</td>
+                            <td>to 50 μL</td>
+                        </tr>
+                        </tbody>
+                    </table>
+                    <p><small>Usually 100 pg – 1 ng of template DNA is sufficient</small></p>
+                    <li>Mix by pipetting up and down or flicking the reactions
+                    <li>Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Step</th>
+                            <th>  </th>
+                            <th>Temperature</th>
+                            <th>Time</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>Initial Denaturation</td>
+                            <td>   </td>
+                            <td>95°C</td>
+                            <td>30 seconds</td>
+                        </tr>
+                        <tr class="even">
+                            <td>25 – 35 cycles</td>
+                            <td>Denaturation</td>
+                            <td>95°C</td>
+                            <td>15 – 30 seconds</td>
+                        </tr>
+                        <tr>
+                            <td>  </td>
+                            <td>Annealing</td>
+                            <td>45 – 68°C</td>
+                            <td>15 – 60 seconds</td>
+                        </tr>
+                        <tr class="even">
+                            <td>   </td>
+                            <td>Extension</td>
+                            <td>68°C</td>
+                            <td>1 minutes per kb</td>
+                        </tr>
+                        <tr>
+                            <td>Final Extension</td>
+                            <td>   </td>
+                            <td>68°C</td>
+                            <td>5 minutes</td>
+                        </tr>
+                        <tr class="even">
+                            <td>Hold</td>
+                            <td>   </td>
+                            <td>4°C </td>
+                            <td>  </td>
+                        </tr>
+                        </tbody>
+                    </table>
+                 </ol>
+            </section>
-                <div>
+<!-- Q5 PCR -->
-                    <i class="fa fa-envelope"></i>
+            <section id="Q5pcr" class="panel">
-                     <p><a href="mailto:iGEM2015dreamteam@groupes.epfl.ch">Contact us</a></p>
+                <a href="https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491"><h1>Q5 PCR</br><small><u>NEB Protocol</u></small></h1></a>
-                 </div>
+                <h2>Materials</h2>
+                <ul>
+                     <li>5X Q5 Reaction Buffer</li>
+                    <li>dNTPs 10µM</li>
+                    <li>Forward and Reverse Primers</li>
+                    <li>Template plasmid DNA</li>
+                    <li>Q5 High Fidelity DNA polymerase</li>
+                    <li>Nuclease-free Water</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Component</th>
+                            <th>25 μL reaction</th>
+                            <th>50 μL reaction</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>5X Q5 Reaction Buffer</td>
+                            <td>5 μL</td>
+                            <td>10 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>10 mM dNTPs</td>
+                            <td>0.5 μL</td>
+                            <td>1 μL</td>
+                        </tr>
+                        <tr>
+                            <td>10 μM Forward Primer</td>
+                            <td>1.25 μL</td>
+                            <td>2.5 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>10 μM Reverse Primer</td>
+                            <td>1.25 μL</td>
+                            <td>2.5 μL</td>
+                        </tr>
+                        <tr>
+                            <td>Template plasmid DNA</td>
+                            <td>1 < 1,000 ng</td>
+                            <td>1 < 1,000 ng</td>
+                        </tr>
+                        <tr class="even">
+                            <td>Q5 DNA Polymerase</td>
+                            <td>0.25 μL</td>
+                            <td>0.5 μL</td>
+                        </tr>
+                        <tr>
+                            <td>Nuclease-free Water</td>
+                            <td>to 25 μL</td>
+                            <td>to 50 μL</td>
+                        </tr>
+                        </tbody>
+                    </table>
+                    <p><small>Usually 100 pg – 1 ng of template DNA is sufficient</small></p>
+                    <li>Mix by pipetting up and down or flicking the reactions
+                    <li>Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Step</th>
+                            <th>  </th>
+                            <th>Temperature</th>
+                            <th>Time</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>Initial Denaturation</td>
+                            <td>   </td>
+                            <td>98°C</td>
+                            <td>30 seconds</td>
+                        </tr>
+                        <tr class="even">
+                            <td>25 – 35 cycles</td>
+                            <td>Denaturation</td>
+                            <td>98°C</td>
+                            <td>5 – 10 seconds</td>
+                        </tr>
+                        <tr>
+                            <td>  </td>
+                            <td>Annealing</td>
+                            <td>50 – 72°C</td>
+                            <td>10 – 30 seconds</td>
+                        </tr>
+                        <tr class="even">
+                            <td>   </td>
+                            <td>Extension</td>
+                            <td>72°C</td>
+                            <td>20 - 30 seconds per kb</td>
+                        </tr>
+                        <tr>
+                            <td>Final Extension</td>
+                            <td>   </td>
+                            <td>72°C</td>
+                            <td>2 minutes</td>
+                        </tr>
+                        <tr class="even">
+                            <td>Hold</td>
+                            <td>   </td>
+                            <td>4°C </td>
+                            <td>  </td>
+                        </tr>
+                        </tbody>
+                    </table>
+                 </ol>
+            </section>
-             </div>
+<!-- PCR PURIFICATION -->
+             <section id="pcrpurification" class="panel">
+                <a href="https://www.qiagen.com/ch/resources/resourcedetail?id=390a728a-e6fc-43f7-bf59-b12091cc4380&lang=en"><h1>PCR Product Purification</br><small><u>QIAGEN Protocol</u></small></h1></u>
+                <h2>Materials</h2>
+                <ul>
+                    <li>PCR products</li>
+                    <li>QIAquick PCR Purification Kit</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Add 5 volumes PB buffer to 1 volume of PCR product and mix</li>
+                    <li>Place QIAquick column in 2 ml collection tube</li>
+                    <li>Apply samples to QIAquick column and centrifuge for 30 – 60 seconds, discard flow-through</li>
+                    <li>Wash by adding 750 μL PE buffer to QIAquick column and centrifuge fo 30 – 60 seconds, discard flow-through</li>
+                    <li>Centrifuge QIAquick column for 1 minutes to remove residual wash buffer</li>
+                    <li>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</li>
+                </ol>
+            </section>
+        </section>
-            <div class="footer-right">
-                <p class="footer-company-about">
+<!-- RESTRCTION DIGEST-->
-                     <span>About us</span>
+            <section id="restriction" class="panel">
-                     Team of a dozen biotechnology students that will attempt to change the world
+                <a href="https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions"><h1>Restriction Digest</br><small><u>NEB Protocol</u></small></h1></a>
-                 </p>
+                <h2>Materials</h2>
+                <ul>
+                     <li>Restriction Enzyme(s)</li>
+                     <li>DNA</li>
+                    <li>10X NEBuffer (Appropriate buffer for used enzyme)</li>
+                    <li>Water</li>
+                 </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Prepare following reaction in 0.5 mL PCR tubes, adding enzyme(s) last:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Component</th>
+                            <th>20 μL reaction</th>
+                            <th>50 μL reaction</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>Restriction enzyme(s)</td>
+                            <td>1 μL (for each enzyme)</td>
+                            <td>1 μL (for each enzyme)</td>
+                        </tr>
+                        <tr class="even">
+                            <td>DNA</td>
+                            <td>100 ng - 1 μg</td>
+                            <td>100 ng - 1 μg</td>
+                        </tr>
+                        <tr>
+                            <td>10X NEBuffer</td>
+                            <td>2 μL</td>
+                            <td>5 μL</td>
+                        </tr>
+                        <tr  class="even">
+                            <td>Water</td>
+                            <td>to 20 μL</td>
+                            <td>to 50 μL</td>
+                        </tr>
+                        </tbody>
+                    </table>
+                    <p><small>20 μL reactions are sufficient for restriction enzyme analysis, larger volumes are usefull if product is used for cloning</small></p>
+                    <li>Incubate at temperature and for duration appropriate for used enzyme (typically 37°C for 15 minutes or 1 hour)</li>
+                    <li>Optional: Inactivate enzyme by incubating reaction at temperature and for duration appropriate for used enzyme (typically 65°C for 20 minutes)</li>
+                </ol>
+            </section>
-                <div class="footer-icons">
+<!--S. CEREVESIAE PROTCOLS-->
+        <section id="yeast" class="panel">
+            <h1>S. cerevisiae Specific Protocols</h1>
-                    <a href="https://www.facebook.com/pages/IGEM-EPFL-2015/1598472927106907" target="_blank"><i class="fa fa-facebook"></i></a>
+<!-- AMINO ACID SOLUTION -->
-                     <a href="https://instagram.com/igem2015epfl/" target="_blank"><i class="fa fa-instagram"></i></a>
+            <section id="aminoacidsolution" class="panel">
-                     <a href="https://twitter.com/EPFL_iGEM/" target="_blank"><i class="fa fa-twitter"></i></a>
+                <h1>Amino acid solution</h1>
-                 </div>
+                <h2>Materials</h2>
+                <ul>
+                     <li>Histidine-Hcl</li>
+                    <li>Uracil</li>
+                    <li>Leucine</li>
+                    <li>Tryptophan</li>
+                </ul>
+                <h2>Procedure</h2>
+                <table cellspacing='0' style="width:90%">
+                    <thead>
+                    <tr>
+                        <th>Stock concentration</th>
+                        <th>Final concentration</th>
+                        <th>Total quantity for 50 mL</th>
+                    </tr>
+                    </thead>
+                    <tbody>
+                    <tr>
+                        <td>100 mM Histidine-Hcl (209 g/mol)</td>
+                        <td> 20.9 g/L</td>
+                        <td> 1.045 g</td>
+                    </tr>
+                    <tr class="even">
+                        <td>20 mM Uracil (112 g/mol)</td>
+                        <td> 2.24 g/L</td>
+                        <td> 0.112 g</td>
+                     </tr>
+                    <tr>
+                        <td>100 mM Leucine (131 g/mol)</td>
+                        <td> 13.1 g/L</td>
+                        <td> 0.655 g</td>
+                    </tr>
+                    <tr class="even">
+                        <td>40 mM Tryptophan (204 g/mol)</td>
+                        <td> 8.16 g/L</td>
+                        <td> 0.408 g</td>
+                    </tr>
+                </tbody>
+                </table>
+                <ol>
+                    <li>Filter and sterilize solutions</li>
+                    <li>Add 8 mL per liter of selective medium or spread 500 μL on a selective plate</li>
+                 </ol>
+            </section>
-             </div>
+             <!-- Integration -->
+            <section id="integration" class="panel">
+                <h1>Yeast integration</h1>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Start a pre-culture of 50mL YPD or SD + Glucose with one colony. Growing at 30°C with shaking (250 rpm) for 16h to 18h untill stationary phase, OD600 > 1,5.</li>
+                    <li>Seed yeast in 500 mL YPD or SD + Glucose. OD should be 0,2. Grow at 30°C in shaker for 3 hours untill OD 0,4 - 0,6.</li>
+                    <li>Spin yeast down 5 min at 800 x g (1800 rpm approx).</li>
+                    <li>Pour off medium and check that the medium is clear (if not, you may have a bacterial contamination).</li>
+                    <li>Wash yeast with 25 mL of water (dH2O). Resuspend and pool cells into one tube.</li>
+                    <li>Spin yeast down, 5 min at 1800 rpm. Decant supernatant.</li>
+                    <li>Resuspend yeasts in freshly prepared TE/LiAc solution (approx 25 mL)</li>
+                    <li>Spin yeast down, 5 min at 1800 rpm. Decant supernatant.</li>
+                    <li>Resuspend yeast in TE/LiAc solution according to the following formula: OD600 x culture volume/60 = vol in which to resuspend (around 2 mL)</li>
+                    <li>Add 10% vol of ssDNA (boiled at 100°C for 5 min and cooled on ice).</li>
+                    <li>Add linearized vectors to the yeast (1 μg).</li>
+                    <li>Add 1 ml of TE/lithium acetate/PEG solution, and resuspend carefully.</li>
+                    <li>Incubate 30 min at 30°C with shaking 200 rpm.</li>
+                    <li>Heat-shock the cells for exactly 20’ at 42°C (in a water bath).</li>
+                    <li>Centrifuge the tubes for 5’’ in a microfuge at full speed at room temperature; remove the supernatant</li>
+                    <li>Resuspend the cells in 50µl of sterile water, and plate onto the appropriate selective media plates using glass beads. Need 2-3 days to grow, at 30°C.</li>
+                </ol>
+            </section>
-         </footer>
+            <!-- PEG LIAC SOLUTION -->
+            <section id="pegliac" class="panel">
+                <h1>PEG/LiAc Solution</h1>
+                <h2>Materials</h2>
+                <ul>
+                    <li>50% PEG  (Polyethylene glycol) prepared with sterile deionized water</li>
+                    <li>10X TE buffer: 0.1 M Tris-Hcl, 10 mM EDTA, ph 7.5, autoclaved</li>
+                    <li>10X LiAc: 1 M lithium acetate, pH 7.5 adjusted with dilute acetic acid, autoclaved</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Prepare PEG/LiAc solution as follows:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Stock concentration</th>
+                            <th>Final concentration</th>
+                            <th>Total quantity for 10 mL solution</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>50% PEG</td>
+                            <td>40% PEG</td>
+                            <td>8 mL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>10X TE buffer</td>
+                            <td>1X TE buffer</td>
+                            <td>1 mL</td>
+                        </tr>
+                        <tr>
+                            <td>10X LiAc</td>
+                            <td>1X LiAc</td>
+                            <td>1 mL</td>
+                        </tr>
+                        </tbody>
+                    </table>
+                </ol>
+            </section>
+            <!-- SD MEDIUM -->
+            <section id="sdmedium" class="panel">
+                <h1>Sd Medium</h1>
+                <h2>Materials</h2>
+                <ul>
+                    <li>Amino Acid Powder</li>
+                    <li>Yeast Nitrogen Base</li>
+                    <li>Ammonium Sulphate</li>
+                    <li>Adenine Sulphate</li>
+                    <li>Water</li>
+                    <li>NaOH</li>
+                    <li>Agar</li>
+                    <li>Glucose</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Place stirrer bar in 2 L Erlenmeyer</li>
+                    <li>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</li>
+                    <li>Adjust pH to 5.9 by adding a few drops of 10 M NaOH</li>
+                    <li>In an other Erlenmeyer, add 35 g agar and 900 mL water</li>
+                    <li>Autoclave both bottles</li>
+                    <li>Transfer the content of first bottle to the agar-containing bottle</li>
+                    <li>Cool to 55°C</li>
+                    <li>Add 100 ml 40% glucose and 16 ml of the required amino acids</li>
+                    <li>Pour plates</li>
+                </ol>
+            </section>
+<!-- YPD MEDIUM -->
+            <section id="ypdmedium" class="panel">
+                <a href="http://www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17602"><h1>ypd medium</br><small><u>Clontech Protocol</u></small></h1></a>
+                <h2>Procedure</h2>
+                <li>Prepare YPD mixture as follows : </li>
+                <ul>
+                    <li>20 g/L peptone</li>
+                    <li>10 g/L Yeast extract</li>
+                    <li>20 g/L Agar (for plates only)</li>
+                    <li>[Optional] For adenine-supplemented YPD (YPDA), add 15 ml of a 0.2% adenine hemisulfate solution per liter of medium (final concentration is 0.003%, in addition to the trace amount of Ade that is naturally present in YPD). Adenine hemisulfate tolerates autoclaving.</li>
+                    <li>Add H2O to 950 ml. Adjust the pH to 6.5 if necessary, then autoclave. Allow medium to cool to ~ 55°C and then add dextrose (glucose) to 2% (50 ml of a sterile 40% stock solution). Adjust the final volume to 1 L if necessary.</li>
+                    <li>[Optional] For kanamycin-containing medium, prepare YPD or YPDA as above. After autoclaved medium has cooled to 55°C, add 0.2–0.3 ml of 50 mg/ml kanamycin (final concentration 10–15 mg/L)</li>
+                </ul>
+            </section>
+         </section>
+<!--SITE DIRECTED MUTAGENESIS-->
+            <section id="mutagenesis" class="panel">
+                <a href="https://www.neb.com/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-protocol-e0554"><h1>Site-directed mutagenesis</br><small><u>NEB Protocol</u></small></h1></a>
+                <h2>Materials</h2>
+                <ul>
+                    <li>Q5 Hot Start High-Fidelity 2X Master Mix (NEB)</li>
+                    <li>Forward and reverse primers</li>
+                    <li>Template DNA</li>
+                    <li>Nuclease-free water</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Prepare following reaction in 0.5 mL PCR tubes on ice, adding Mater Mix last:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Component</th>
+                            <th>25 μL reaction</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>Q5 Hot Start High-Fidelity 2X Master Mix</td>
+                            <td>12.5 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>10 μM Forward Primer</td>
+                            <td>1.25 μL</td>
+                        </tr>
+                        <tr>
+                            <td>10 μM Reverse Primer</td>
+                            <td>1.25 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>Template DNA (1-25 ng/μL)</td>
+                            <td>1 μL</td>
+                        </tr>
+                        <tr>
+                            <td>Nuclease-free Water</td>
+                            <td>9 μL</td>
+                        </tr>
+                        </tbody>
+                    </table>
+                    <li>Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Step</th>
+                            <th>  </th>
+                            <th>Temperature</th>
+                            <th>Time</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>Initial Denaturation</td>
+                            <td>   </td>
+                            <td>98°C</td>
+                            <td>30 seconds</td>
+                        </tr>
+                        <tr class="even">
+                            <td>25 cycles</td>
+                            <td>Denaturation</td>
+                            <td>98°C</td>
+                            <td>10 seconds</td>
+                        </tr>
+                        <tr>
+                            <td>  </td>
+                            <td>Annealing</td>
+                            <td>50 – 72°C</td>
+                            <td>10 – 30 seconds</td>
+                        </tr>
+                        <tr class="even">
+                            <td>   </td>
+                            <td>Extension</td>
+                            <td>72°C</td>
+                            <td>20 - 30 seconds per kb</td>
+                        </tr>
+                        <tr>
+                            <td>Final Extension</td>
+                            <td>   </td>
+                            <td>72°C</td>
+                            <td>2 minutes</td>
+                        </tr>
+                        <tr class="even">
+                            <td>Hold</td>
+                            <td>   </td>
+                            <td>4°C </td>
+                            <td>  </td>
+                        </tr>
+                        </tbody>
+                    </table>
+                    <li>Assemble following reagents:</li>
+                    <table cellspacing='0' style="width:75%">
+                        <thead>
+                        <tr>
+                            <th>Component</th>
+                            <th>Volume</th>
+                        </tr>
+                        </thead>
+                        <tbody>
+                        <tr>
+                            <td>PCR Product</td>
+                            <td>1 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>2X KLD Reaction Buffer</td>
+                            <td>5 μL</td>
+                        </tr>
+                        <tr>
+                            <td>10X KLD Enzyme Mix</td>
+                            <td>1 μL</td>
+                        </tr>
+                        <tr class="even">
+                            <td>Nuclease-free Water</td>
+                            <td>3 μL</td>
+                        </tr>
+                        </tbody>
+                    </table>
+                    <li>Mix by pipetting up and down and incubate at room temperature for 5 minutes</li>
+                    <li>Thaw completent E. coli cells on ice</li>
+                    <li>Add 5 μL of KLD mix to the tube of thawed cells. Flick tube 4-5 times to mix.</li>
+                    <li>Place mixture on ice for 30 minutes</li>
+                    <li>Heat shock at 42°C for 30 seconds</li>
+                    <li>Place on ice for 5 minutes</li>
+                    <li>Pipette 950 μL of room temperature SOC into the mixture</li>
+                    <li>Incubate at 37°C for 60 minutes with shaking</li>
+                    <li>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</li>
+                </ol>
+            </section>
+<!-- T4 LIGATION -->
+                        <section id="t4ligation" class="panel">
+                            <h1>Rapid ligation using T4 ligase</h1><br><small> Based on Invitrogen protocol (for cohesive ends)</small></h1>
+                            <h2>Materials</h2>
+                            <ul>
+                                <li>5X ligase reaction buffer</li>
+                                <li>vector DNA</li>
+                                <li>insert DNA</li>
+                                <li>autoclaved distilled water</li>
+                                <li>T4 DNA ligase</li>
+                            </ul>
+                            <h2>Procedure</h2>
+                            <ol>
+                                <li>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</li>
+                                <li>Mix gently. Centrifuge briefly to bring the contents to the bottom of the tube</li>
+                                <li>Incubate at room temperature for 5 min</li>
+                                <li>Use 2 μl of the ligation reaction to transform competent cells</li>
+                            </ol>
+                        </section>
+<!-- TAE BUFFER -->
+            <section id="tae" class="panel">
+                <h1>Tris-Acetate-EDTA (TAE) buffer 50X</h1>
+                <h2>Materials</h2>
+                <ul>
+                    <li>Acetate 100%</li>
+                    <li>Tris base</li>
+                    <li>EDTA 0,5M</li>
+                </ul>
+                <h2>Procedure (1L)</h2>
+                <ol>
+                    <li>Add 100mL of 0,5M EDTA (pH 8.0)</li>
+                    <li>Add 242g of Tris base </li>
+                    <li>Add 57,1mL of glacial acetic acid (100%)</li>
+                    <li>Fill up to 1L with water (adjust pH to ~8.3)</li>
+                    <li>Send to autoclave</li>
+                    <p><small>To prepare 1L of 1X TAE dilute 20mL of 50X TAE in 980mL of water.</small></p>
+                </ol>
+            </section>
+<!-- TRANSFORMATION -->
+            <section id="transformation" class="panel">
+                <a href="https://www.neb.com/protocols/2012/12/11/gibson-assembly-transformation-protocol-e5510"><h1>Transformation</br><small><u>NEB Protocol</u></small></h1></a>
+                <h2>Materials</h2>
+                <ul>
+                    <li>Competent cells</li>
+                    <li>DNA</li>
+                    <li>SOC medium (SOB + Glucose)</li>
+                    <li>Petri dish with appropriate antibiotic resistance</li>
+                </ul>
+                <h2>Procedure</h2>
+                <ol>
+                    <li>Thaw competent cells on ice</li>
+                    <li>Add 2 μL DNA to the competent cells, mix by pipetting up and down or flicking the tube 4-5 times</li>
+                    <li>Place mixture on ice for 30 minutes</li>
+                    <li>Heat shock at 42°C for 30 seconds</li>
+                    <li>Transfer tubes to ice for 2 minutes</li>
+                    <li>Add 950 μL room-temperature SOC media</li>
+                    <li>Incubate at 37°C for 60 minutes with shaking</li>
+                    <li>Spread 100 μL cells onto selection plates (warm plates to 37°C prior to this step for increased efficiency)</li>
+                    <li>Incubate overnight at 37°C</li>
+                </ol>
+            </section>
+        </div>
+    </div>
+</div>
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Difference between revisions of "Team:EPF Lausanne/Notebook/Protocols"

Latest revision as of 23:45, 16 September 2015

Protocols

Agarose Gel

Materials

Procedure

Competent Cell PreparationOpen Wet Ware Protocol

Materials

Procedure

5' DephosphorylationNEB Protocol

Materials

Procedure

Gel extractionQIAGEN protocol

Materials

Procedure

Gibson AssemblyNEB Protocol

Materials

Procedure

Glycerol StockAddgene Protocol

Materials

Procedure

Lysogeny Broth (LB) Medium

Materials

Procedure (for 1 L)

Lysogeny Broth (LB) Agar Plates

Materials

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

MiniprepQIAGEN Protocol

Materials

Procedure

Comments

Polymerase Chain Reaction (PCR)

Colony PCR

Materials

Procedure

Phusion PCRNEB Protocol

Materials

Procedure

Taq PCRNEB Protocol

Materials

Procedure

Q5 PCRNEB Protocol

Materials

Procedure

PCR Product PurificationQIAGEN Protocol

Materials

Procedure

Restriction DigestNEB Protocol

Materials

Procedure

S. cerevisiae Specific Protocols

Amino acid solution

Materials

Procedure

Yeast integration

Procedure

PEG/LiAc Solution

Materials

Procedure

Sd Medium

Materials

Procedure

ypd mediumClontech Protocol

Procedure

Site-directed mutagenesisNEB Protocol

Materials

Procedure

Rapid ligation using T4 ligase

Materials

Procedure

Tris-Acetate-EDTA (TAE) buffer 50X

Materials

Procedure (1L)

TransformationNEB Protocol

Materials

Procedure

Competent Cell Preparation
Open Wet Ware Protocol

5' Dephosphorylation
NEB Protocol

Gel extraction
QIAGEN protocol

Gibson Assembly
NEB Protocol

Glycerol Stock
Addgene Protocol

Miniprep
QIAGEN Protocol

Phusion PCR
NEB Protocol

Taq PCR
NEB Protocol

Q5 PCR
NEB Protocol

PCR Product Purification
QIAGEN Protocol

Restriction Digest
NEB Protocol

ypd medium
Clontech Protocol

Site-directed mutagenesis
NEB Protocol

Transformation
NEB Protocol