Digestion Round 2

Digestion round 2: Liquid culture 

4mL of enzyme buffer means 4 batches

1. Spin aspergillus liquid culture until network is finely broken up

2. Using sterile syringe, placed 11mL into 15mL tube

3. Spin liquid culture at 2500 RPM for 10 minutes**

4. Pipetted 1mL of enzyme buffer solution into 4 tubes

5. Pipetted 1mL of ONLY buffer solution into 1 tube

6. Pipetted 1mL of spun LC into all tubes**

7. Digestion

**Pipette tips are too small to get 1mL and you get clogging. I ended up squirting the lc back in which disrupted the centrifuge gradient.. So, cut the pipette tips for liquid culture.

Digestion start time: 1:56pm 

Batch #1: Control - Incubated 1:1 lc with 0.6 M MgSO4 - no enzymes

Batch #2: 2hr enzyme digestion - 2hours at 30°C in 80 RPM

Batch #3: 2hr enzyme digestion - 2hours at room temp still 

Batch #4:  Left overnight 

2hrs in the shaking incubator. Then left overnight and filtered at 11:35am the next morning. Rinsed with 5mL nuclease free water and centrifuged for 10 minutes with ~3mL sucrose buffer. 

1. Make 1.2M sucrose cushion for centrifuging 

342.3g/mL 

10.269g/25mL

1. Filter the digestions into 50mL tubes through cell strainer

2. Slowly pipette 3mL ish underneath the filtered fluid

3. Centrifuge for 10 minutes

4. Pipetted 20ul onto slide to look for protoplasts.

5. Videos of three processed batches (#1, #2, #3) can be found here:

Conclusion: Between the control and the digested mycelium, there is definitely ‘stuff’ in the liquid. But nothing convincing. 

Thoughts:

• Enzyme solution should be fresh. It sat overnight and looked kind of murky.

• The sucrose cushion did not seem to have a distinctive line compared to the first time.

• The protocol rinses the strainer… maybe getting any remaining protoplasts into the filtered solution.

• I did not see stuff in that middle area, I think more fungal tissue is a good idea.