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

Protocol provided by Fabiano Jares from DTU Bioengineering. The protocol is for species of Aspergillus. We used it for Aspergillus oryzae.

Materials

• Aspergillus protoplastation buffer (APB) 1L solution

• Final conc: 1.1 M MgSO₄ and 10 mM Na-phosphate buffer. (Hint: use 1 M solutions of Na₂HPO₄ and NaH₂PO₄ to prepare the sodium phosphate buffer). pH is adjusted with 2 N NaOH to 5.8.

• APB with Glucanex (40 mg Glucanex/ml APB)

• Aspergillus transformation buffer (ATB) 1 L solution

• Final conc: 1.2 M Sorbitol; 50 mM CaCl₂·2 H₂O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.

• PCT (200 mL stock solution)

• Final conc: 1.2 M Sorbitol; 50 mM CaCl₂·2 H₂O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.

• Final conc: 50 % w/vol PEG 8000; 50 mM CaCl₂; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store PCT at 4 °C.

Procedure

Protoplastation

1. Collect the conidia from a plate by adding 5 mL of sterile liquid MM (or YPD for A. niger) with required supplements and firmly rubbing the colonies with a sterile Drigalski spatula. The conidial suspension is withdrawn from the plate and added to the shake flask containing 100 mL of media.

2. The culture is incubated over night (or 2 days for A. niger and other strains growing slower) at appropriate temperature and 150 rpm.

3. Harvest the mycelia and germlings by using Mira cloth.

4. Wash the mycelia with Aspergillus protoplastation buffer (APB) to remove the liquid media from the mycelia.

5. Resuspend the mycelium in 10-20 ml APB solution containing 40 mg Glucanex/ml APB. Homogenize mycelial and enzyme suspension gently to obtain the best possible digestion of the fungal cell wall.

6. Shake at the 30°C and 150 rpm for 2-3 hours.

7. Filter through Mira cloth and collect the flow through.

8. Add APB up to the total volume 40 ml.

9. Carefully make an overlay with 5 ml of 2 fold diluted Aspergillus transformation buffer (ATB). Dilute with sterile Milli-Q H2O.

10. Centrifuge at 3000xg (acceleration 9, deceleration 4) for 12 min.

11. Upon a successful protoplastation, a halo of white protoplast slurry is caught just below the surface. Collect protoplast slurry and transfer it to new tube.

12. Add ATB up to the total volume 40 ml.

13. Centrifuge at 3000xg (acceleration 9, deceleration 9) for 12 min. Discard supernatant.

14. Resuspend the protoplasts in approx.. 1 ml ATB.

Transformation

15. Gently mix DNA and protoplast in an eppendorf tube (for a simple transformation 50μL protoplast is enough).

16. Add 150 μL PCT.

17. Mix by inversion of the tube.

18. Incubate 10 min at room temperature.

19. Add 250 μL ATB and mix.

20. Plate on osmotic stabilized, selective media.

Notes

1. The volume of DNA in water should preferably be kept below 25 % of the total volume of DNA-protoplast mix to avoid osmotic stress in protoplasts. The amount of DNA needed for a successful transformation varies with the type of the DNA substrate.

• For linear DNA, add ≈10 μL linearized plasmid

• Use self-replicating plasmids as positive controls to test the competence of the protoplasts and evaluate the success of transformation. AMA1 plasmids (pLAT4-3): add ≈2 μL miniprep.

• Remember to include a negative control plate, where protoplasts are plated without any DNA.