I’m going to need to amend my category system. I feel like it makes very little sense to have it organized the way it is. I’ll have to spend some time thinking about how I want these experiments arranged.
Regardless, I’m doing an extension of the experiments from last week. This time I’m going to compare the growth from a regular LB broth sample, to growth in DDW and D2O. Here is my setup:
- Prepare LB broth in DDW and D2O. It’s 1g in 50ml for the stuff I have (maybe this is standard).
- It is crucial to note that I did not autoclave after dissolution. The reason is because the autoclave is filled with regular water and my fear is that the mystery system that is the autoclave will mix too much water with D2O and depleted D water. So I opted against this for the time being.
- In the future (this is where the #SciFund challenge comes in) I could spend a ton of money just on water so I can fill the autoclave with DDW/D2O to autoclave each of those samples. That would be costly and I would need to find out how much water I need to do one autoclave, and how long LB/YPD is good in storage.
- From the starter culture Alex prepared yesterday, add 1ml of culture to 3 test tubes labeled DDW, D2O, and H2O (for the regular water sample).
- I want 1:10 dilutions of each, so add:
- 9ml of LB-DDW to the DDW test tube
- 9ml of LB-regular to the H2O test tube
- 3ml of LB-D2O and 6mL of LB-DDW to the DDW test tube (because I want 30% D2O – I don’t think the cells would do very well in 90% D2O)
- Take 500ul from each sample and put them in semi-micro cuvettes. Also use 500ml from LB-broth with no cells in it to use for a blank.
- Measure the absorption in your nanodrop.
I’ve already made some mistakes in this experiment:
- There is the autoclave issue. Tomorrow I’ll try again and I’ll autoclave some DDW and D2O broth to see if the growth rates change.
- I ran out of semi-micro cuvettes and so I have to use macro cuvettes because our even smaller micro cuvettes aren’t giving accurate readings. I’m testing various blanks to verify this. This all means that I have to use 1ml of sample for each measurement, which will deplete the cultures in a few hours. Not good
- I also didn’t think to blank each sample with it’s own broth. Ie use the LB-DDW to get a blank measurement for the DDW sample, use LB-D2O for the D2O sample, etc. And I got rid of those broths (well one was used up completely, the other was disposed of).
The one positive is that I did starter cultures is 30% D2O and pure DDW so that I can redo this experiment tomorrow with hopefully much better results. I’ll be preparing the LB broths later today with a quick protocol of that.