For Pfx PCR the reaction mixture is:

PCR program pFX :

For Taq PCR the reaction mixture is:

PCR program Taq polymerase:

*Annealing temperature is very dependent on primer. Optimal temperature: 3x G/C + 2x A/T

• QIAquick columns
• buffer QG
• buffer PE
• isopropanol
• milliQ
• microcentrifuge
• heat block at 50 °C

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µL). For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
   IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH <7.5. Buffer QG contains a pH indicator which is yellow at pH <7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage.
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. The maximum volume of the column reservoir is 800 µL. For sample volumes of more than 800 µL, simply load and spin again.
8. Discard flow-through and place QIAquick column back in the same collection tube.
9. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
10. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 10,000 x g (~13,000 rpm).
11. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13. To elute DNA, add 50 µL of Buffer EB (10 mM Tris•Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.
    Important: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µL from 50 µL elution buffer volume, and 28 µL from 30 µL. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris•Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

• Your favorite yeast strain
• Vybrant DyeCycle Orange from invitrogen
• PBS
• 37°C heat block

1. Centrifuge cells on a table top centrifuge for 10 s on 13.000 rpm
2. Take of supernatant and add PBS
3. Add 2 µL/mL Vybrant DyeCycle Orange and vortex until homogenuous solution
4. Keep for 30 minutes on 37°C and the cells are stained.

• Yeast culture
• Vybrant® DyeCycleTM Orange stain
• Ligand
• Flow cytometry instrument

1. Grow yeast cells at 30C overnight and re-inocculate in 10 mL medium the next morning
2. If cells are in exponential growth phase add the ligand (also keep a control without ligand)
3. After incubation stain the cells according to protocol
4. Transfer the stained cells in a flow cytometry tube
5. Analyze the samples on a flow cytometer using 488 nm excitation or 532 nm excitation and orange emission
6. After each measurement flush the tube with PBS buffer

• Yeast culture
• Ligand
• Flow cytometry instrument

1. Grow yeast cells at 30C overnight and re-inocculate in 10 mL medium the next morning
2. If cells are in exponential growth phase add the ligand (also keep a control without ligand)
3. Transfer the cells in a flow cytometry tube
4. Analyze the samples on a flow cytometer using 488 nm excitation and 509 nm emission
5. After each measurement flush the tube with PBS buffer

• Yeast culture
• Ligand
• Fluorimeter plate reader
• 96 well plate

1. Grow yeast cells at 30C overnight and re-inocculate the next morning
2. Measure after approx. 3h hours the OD600. Dilute the cells with medium until an OD of approx. 0.1
3. Transfer the cells to the 96 well plate. Don't forget to have a well with only medium.
4. Add the ligand.
5. Analyze the samples in the fluorimeter that is kept at 30C. Use filters with 488 nm excitation and 509 nm emission. At the same time measure the OD600.
6. Calculate the fluoresce per biomass by dewing the fluorescence by the OD600 values

• Yeast cultures (wt with and without alpha feromones and far1KO with and without alpha feromones)
• Alpha feromones
• Spectrophotometer
• 30C incubator

1. Grow yeast cells at 30C overnight and re-inocculate 20 ml of medium with 500µl of grown cells the next morning
2. Measure OD600 every hour.
3. Add alpha feromones after 3hr
4. Measure OD600every hour, dilute if needed.

• 4 x Two yeast cultures with each having six yeast-strains
• Ligands
Water 2 x Alpha pheromone 1000x diluted 2 x Isoamyl acetate 99% Methyl phenylacete 99% Niacin 0.336 g/100 ml Methyl nicotinate 10 mM
• Typhoon fluorescence scanner
• 8 Resealable (hard) plastic boxes of 10x10x10 cm.

1. Grow yeast cells at 30C for two days.
2. Analyze the samples in the Typhoon fluorescence scanner before starting experiment.
3. Pore approx. 25 ml of every ligand (except alpha pheromone) in the box after cleaning it with alcohol / autoclaving if possible.
4. Place two sided tape on the inwards part of the boxlid. Gently but firmly push the petri dish on it. Remove the lid of the dish and put the dish upside down in the box. For alpha pheromone; add solution directly on all yeast strains.
5. After 3.5 hours remove the dish from the lid.
6. Analyze the samples in the Typhoon fluorescence scanner. Use filters with 488 nm excitation and 509 nm emission. Set PMT to 300. Orientation of dish to Platen.

If one want to get insight into the real in situ working of genetic circuits of S. Cerevisiae one must look at it at a smaller scale: The cell scale. For this a number of techniques are available like e.g. flow cytometry. One of the drawbacks is that a certain single cell can only be measure once and that it is not possible to do analysis on a single cell in the time dimension. Therefor fluorescence microscopy can be of much help to assess the overtime state dynamics of the single cell. For this certain preparations are needed:

• Fluorescence microscope
• Concanavalin A type V, Sigma-Aldrich
• Yeast cells
• OD medium
• Sterile water

1. Grow yeast cells prior to the experiment for at least 15 hours in DO medium at 30 degrees. For the experiment the cells should be in exponential phase.
2. Incubate a glas bottom black walled 24wells plate with 500µL of Concanavalin A solution (100 µg/ml )for at least one hour.
3. Aspirate liquid by pipetting it out of the corner of the well.
4. Apply a 500µL solution of yeast cells ( OD600=0.1-0.3 ) that has been mildly sonicated and let it incubate for 10 minutes
5. Apply a 500µL solution of yeast cells ( OD600=0.1-0.3 ) that has been mildly sonicated and let it incubate for 10 minutes.
6. Wash 3 times with OD medium to remove unbound cells from the well.
7. Apply OD medium to the well and add ligand of choice.
8. Incubate the cells in a preheated microscope chamber of 30 degrees while performing the experiment.

Since this is the first time that TU Delfts iGEM team is working with yeast, we faced a lot of small yeast-related 'challenges'. With this page we want to inform you about the basics and the pitfalls of working with yeast.

Basics

Yeast, Saccharomyces cerevisiae, is a simple, unicellular eukaryotic organism. This organism has been used for fermentation and baking for over 4000 years and it is probably the oldest domesticated micro-organism in the human history. An important one too, can you imagine a life without bread and beer? (Even if you can imagine it, it would most definitely be less fun!) Nowadays the whole genomic sequence of Saccharomyces cerevisiae is known and a lot of genomic tools are available.

Auxotrophy

The main advantage from an engineering perspective is that yeast has Auxotrophic markers. In specific strains genes are knocked out which synthesize essential enzymes in the amino acid synthesis routes. By complementing these deficiencies by adding the necessary gene on your DNA this provides a nice selection procedure.

PRS huttle vectors

The name pRS415 gives an indication on the presence of a CEN/ARS replication origin. 0 means yeast integrative plasmid, 1 means that it also can be used to maintain the plasmid in circular form. pRS415 Gives an indication of the auxotrophic marker used. pRS415 Version number… not really different.

Chromosomal integration

We encountered a lot of problems with plasmids. Because we wanted our constructs to be universal (with the idea to make it suitable for ‘fast checking’) we tried maintaining a plasmid. As it turned out, yeast cells are not eager to maintain a plasmid and with our construct we suspect homologous recombination occurred. After transformation, a PCR on the transformed plasmid, obtained by isolation, showed two bands instead of the suspected single band, one being ! Integration of the plasmid is therefore advised! Checking of this can be quite gruesome optimizing the necessary PCR reactions on your transformed yeast colonies. Chromosomal isolation can therefore improve the steps.

Knock-out strains

European iGEM teams have the advantage to have Euroscarf available to order strains with knocked out ORFs. The typical nomenclature is also explained here: Euroscarf explanation.