Difference between revisions of "Team:DTU-Denmark/Experiments"

DNA from IDT will typically be delivered in a white flaky substance, which need to be resuspended, before the DNA is ready for use

Materials

• Table centrifuge

• EB/TE buffer

• Genes/Primers from IDT or other DNA provider

Procedure

1. Quickly spin the DNA down in the table centrifuge

2. Calculate the amount of EB buffer need to dilute the gblocks to a desired concentration. Important: gene fragments and primeres are not diluted to the same concentration: The concentration of gene fragments is usually 25 ng/μL and for primers it is 100 μM.

3. Genes/Primers from IDT or other DNA provider

   • DNA from IDT usually comes in dried flakes of 500 or 1000 ng of DNA. To achieve the desired concentration (usually 25 ng/μL) the needed amount of EB buffer is 40 μL (for 1000 ng samples) or 20 μL (for 500 ng samples)

   • In order to calculate the the molar amount of primer, use the NEB calculator

4. Add the calculated amount of EB buffer

5. Store the resuspended DNA at -20°C

Basic protocol on plating of the fungi.

Materials

• Agar plates with media of interest

• Sterile toothpicks

Procedure

Plating using mycelia

1. Open the plate containing the mycelia from the species of interest

2. With a sterile toothpick, scratch the surface of the mycelia.

3. Spike the new agar plate with the toothpick.

4. Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle.

5. Put the plate into a plastic growth bag.

6. Place growing bag into the incubator.

Plating using spores

1. Touch the sterile tooth pick in the spore suspension.

2. Spike the new agar plate with the toothpick.

3. Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle.

4. Put the plate into a plastic growth bag.

5. Place growing bag into the incubator.

Minimal media used for protoplastation of A. oryzae. This is for 1 L of media.

Materials

• 50 mL D-glucose

• 50 mL Nitrate salts

• 1 mL Trace elements

• 1 mL Thiamine

• 20 g Agar (SO.BI.GEL)

Procedure

Mix

1. Mix in a blue cap flask.

2. Add water until the volume is 1 L.

3. Autoclave.

Minimal medium: Schizophyllum commune SMM agar (1L) needed for S.commune plates

Materials

• Glucose - 20g

• Monopotassium phosphate KH₂PO₄ - 0.46g

• Potassium phosphate dibasic K₂HPO₄ 3H₂O - 1.28g

• Magnesium sulfate MgSO₄ 7H₂O - 0.5g

• Trace elements solution - 1ml

• FeCl₃ solution - 1ml

• L- Asparagin - 1.5g

• Agar - 20g

• Thiamine (10mg/100ml) - 1.2ml

Procedure

Preparation of the media

1. Weigh and add the required solid components (except agar)

2. Add the required liquid components in a bottle

3. Dissolve in demineralized water until the total volume is 900ml

4. Stir the liquid using a magnet (no pH adjustment is required)

5. Add the required quantity of agar

6. Add demineralized water until the desired total volume (1L)

7. Autoclave

8. After sterilization add 1.2 ml of filter sterilized thiamine