LAB3 - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

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material#LAB3

Summary:

• Analyze PCR products from LAB2 on gel#2
• Inoculate S. cerevisiae in liquid YPD cultures with from LAB2

It is critical that you keep track of your PCR tube. They are very small and have only a number written on them. We need them for the transformation later, so we can not lose them.

1. Put the gel in the gel tray in a square petri dish. The gel should have at least one well per group number and one extra for the marker.
2. Add 5 µL of the PCR product DNA to an empty well, start with the leftmost well.
3. All group members should load their PCR product.
4. Take note of where your PCR product is.
5. The teacher will help you to put the gel in the electrophoresis chamber.
6. The teacher will then load the molecular weight marker.
7. Apply the electrical field as soon as you are done.
8. The electrophoresis last around 15 min at 200 volts in the Bachman gel tank using a homemade rectifier.
9. When the gel run is completed, the teacher with take a picture using a transilluminator.

The first task is to measure the optical density of the culture. We do this by diluting the culture ten times in the same medium and measuring the OD640 nm against the medium as blank.

1. Add some YPD medium to a plastic weighing boat. This medium does not need to be sterile.
2. Add 900 µL YPD from the weighing boat to a cuvette.
3. Mix cell culture and add 100 µL to the cuvette.
4. Measure OD640 with a spectrophotometer.
5. Calculate the optical density of the culture.
6. Calculate the volume of cells from the pre-culture that needs to be added to 40 mL of YPD medium to attain an OD640 = 0.17 (OD640 = 0.17 = 5 x 106 cells/ml using a spectrophotometer GENESYS20.)

We can calculate the culture volume we need to add by the equation below. This is a simple mass balance:

$Va \cdot OD640 = 0.17 \cdot (40 + Va)$

• Va is the volume we use to inoculate (mL).
• OD640 is the optical density for the the culture from which we inoculate.
• The culture volume before inoculating is 40 mL.
• The final optical density we want is 0.17.

If we rearrange the equation: $Va = \frac{6.80}{OD640 - 0.17}$

1. Wipe down the bench with ethanol and light the lamp to create a sterile environment.
2. Add 40 mL of YPD medium to a 50 mL FALCON tube.
3. Add $Va$ mL of pre-culture to your tube.
4. Mix and measure the OD640 by removing one mL to an empty cuvette
5. If the OD640 is ok, pour all the contents of the tube into a sterile Erlenmeyer flask.
6. Incubate until cells have grown for two generations i.e. final OD640 should be 0.17 * 2 * 2 = 0.68
7. Pour the cells into a sterile 50 mL FALCON tube and store on ice.

Each group should make 500 mL solid SD medium in a 1 L Schott flask. Put a small piece of autoclave tape on the lid. Label the flask properly, (Content, Date, Course, Turno, Group etc.).

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