Category Archives: D2O adaptation

YPD setup and Starter Culture prep

This is going to be a hyper detailed post because I’m unable to work in the lab today (soccer injury) and so Steve will be filling in. So pardon me while I write the tiniest of details about the lab so Steve can access everything I need him to:

Making YPD:

  1. Supplies:
    1. You will need 3 beakers. The beakers are stored above the autoclave and there may be more in the cabinet below and to the right of the sink. You’ll want beakers that can hold well over 100ml because when you stir you may spill some.
    2. You will also need 3 bottles with caps for long term storage of the prepared YPD. Those are also above the autoclave.
    3. While you are here, the aluminum foil is in the drawer under the autclave, you’ll need some.
    4. The hotplate stirrer is in the front of the lab near the sink and microcentrifuge.
    5. YPD broth mixture is on the wet lab bench with all the powder chemicals right behind Kiney’s computer.
    6. The scale is in the front of the lab next to the PCR machine, and there are mini weigh boats around there too (aolong with regular sized weigh boats).
    7. Scoops and scoopulas are near the sink in the back. There are two cups one is labeled for dirty scoops and the other for clean. You’ll need a clean one.
    8. Medium gloves are in the top left most drawer opposite the autoclave (and under the seed growth station).
    9. Syringes and syringe filters are in the right most lower cabinet opposite the Kiney computer. There are some really big syringes (60ml I think) and filters. Use 1 filter per squeeze.
    10. Stir bars are magnetized to the bench right about eye level near the powder chemicals. You’ll need three, and you might as well grab a magnet too so you can get the stirbars back.
    11. New bottles of DDW are kept in the desicator next to Nadia’s bench. New bottles of D2O are kept next to the powder chemicals. You will need one of each.
    12. Sigma DI bottled water is above the seed growth station. You will need 100ml of this.
    13. The autopipetter is somewhere in the lab. That thing gets around quite a bit. It is either somewhere around Nadia’s bench, somewhere in the front of the lab (maybe in it’s holder near the pipette tips), or near my bench hanging above or near the laptop. Tips are in the front of the lab next to the peeper PC.
    14. Sharpie – these things are everywhere in the lab. If you can’t find one there should be like 3 sitting on my bench.
  2. Measure the amount of DDW and D2O in each bottle and put in beakers. Cover with aluminum foil. And measure 100ml of DI water and put into the third beaker.
  3. Calculate the amount of YPD needed based on what it says on the YPD bottle. From this post here, it looks like you need 5g per 100ml of water. And the D2O should yield about 90ml of water.
  4. Add the ypd to the beakers with water (and make sure you keep track of which beaker has which water) and one by one, using the stirrer, mix until dissolved.
  5. Filter this water into the bottles. It takes kinda a while to get full dissolving so while one is mixing you can syringe and filter an already stirred water into a bottle.
  6. Label the water type.

You’ve just made YPD in different water types! Yay! Ok now to make the starter cultures.

  1. Gather supplies:
    1. yeast – in the fridge on the right side (and maybe near the bottom) is a rack of PCR tubes with yeast starters in it. It should say yeast on the caps, but it may be hard to read.
    2. test tubes – are above the autoclave in a blue test tube holder. There should be 2 holders and one should be empty. You’ll need 3 test tubes.
    3. prepared ypd – you just made this!
    4. disposable inoculation loops – these are in the drawer under the laptop. There are two colors, green and blue and each is a different size. I can’t remember which one is smaller, but you’ll need the smaller one because the larger one won’t fit in the PCR tube.
    5. ypd plates – might as well streak some yeast while we’re at it. These are on the top most part of the bench above the seed growth station. Take out one or two if you please.
    6. aluminum foil – again, since you just used this it should be accessible to you.
    7. incubator/shaker – in the chase near the hole in the lab, where the banana picture was made
  2. Put 10ml of each water type into a test tube and label it. Sharpies abound on my bench so you should be able to find one.
  3. Use the inoculating loop to inoculate the yeast in the liquid media. One loop per tube and swoosh around! Fun right?! (BTW, the ?! combo is called an interrobang and there is an interesting wikipedia article on this.)
  4. With the ypd plate, streak some cells from the starter tube onto here. And since there will be a lot of liquid left in the tube, I like to just poor the tube onto another ypd plate to ensure something starts growing. Paranoia is a bitch.
  5. Bring your new colonies to the incubator, make sure the incubator is set for 30C and about 100rpm. According to this post I set it for 125rpm because I can.

Yay! You have made fire! Now just leave it til tomorrow when hopefully I can return to do the next phase of experiments.