User:Anthony Salvagno/Notebook/Research/2009/02/05/Spheroplasting Try 1

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quick notes to be formatted later

made 2 yeast cultures. incubated over night. made 4 samples from those 2. starting OD's .892 and .802. split into 4 groups (2 each) and took time points to monitor enzyme (lyticase) reaction. when completed added proteinase K to end chewing.

Goal of Today

The object of today was to prepare the cells for spheroplasting and then removal of everything not DNA/RNA. We had to make a few on the fly alterations to the protocol but in the end everything worked out well. For the entire DNA prep protocol please visit User:Anthony Salvagno/Notebook/Research/Yeast Genomic DNA Prep.

We focused on steps 5-15 of the protocol. See notes below.

Notes

Precursor

  1. Measure OD 600nm
  2. Dilute to OD=0.0005 in 25mL YPD
    • Store in 30C overnight

Kelly did these steps last night for me. He took some notes as follows:

  • The initial OD measured was 0.958
  • To achieve the approximate OD needed (0.5 by morning) he diluted 13uL in 25mL YPD.
  • Kelly took the liberty of creating a second batch from our initial batch (same everything).
  • He finished at 6:30pm.
  • Allowed to incubate overnight.

Today

Below are the steps we did today and I will provide my comments as subpoints to each step.

can excel save files as images instead of this ugly printscreen?
  1. Measure OD (hopefully will be around .5)
    • We started at around 9:10 this morning. Batches labeled 1 and 2.
    • Initial OD's were 0.892 for batch 1 and 0.802 for batch 2.
    • Since there was 25mL of yeast for each batch, we decided it was best to make 2 batches from each batch (labeled x.y, ie batch 1 from batch 1 was labeled 1.1 in my notes, batch 1 from batch 2 is labeled as 2.1, etc.)
  2. Spin 10 ODs of cells
    • Used table top centrifuge which spins at 4500 rpm for 2 min (default). We let it spin for 1 min.
    • Next we poured all the excess liquid out and all that remained was pellet of cells.
  3. Resuspend pellet in 5mL of sheroplasting buffer (SB) with 50mM 2-ME and 150uL lyticase. Incubate on ice.
  4. monitor OD of 1/4 dilutions in 1% SDS (let sit in SDS for 2 min before measuring). OD should drop from .5 to < .1 over about 30 min
    • we took 5 time points, see graph on right for plot and data
    • when measuring OD we added 750uL SDS to each spectroscopy tube, but for the control we put in 750uL SDS and 250uL SB (which is what gets added to yeast cells).
    • After about 60 min we added an extra 100uL lyticase to speed up the reaction.
    • This took way longer than the estimated 30 min, but the extra addition of lyticase really sped up the process (see graph).
    • The lyticase is an enzyme which is supposed to break down the cell wall of the yeast. If given enough time, the lyticase will rupture the cells and destroy more than just the cell wall. I got to see the end product under a microscope. When given the perfect amount of time (our final version) the cells were all very round, and there were a few that looked like broken eggs (with goop all hanging out the sides).
  5. Add 5mL Proteinase K Buffer (PK; to be made fresh) Proteinase K Buffer Protocol
    • we added 4mL because after all the time points, we only had 4mL of yeast cells left. Kelly said we only needed a volume for volume amount.
  6. Incubate for 30 min at 65C
  7. Add 2mL 5M KOAc
    • Due to solubilities of stuff, the KOAc was able to help disassociate the parts of the cells we don't want from the parts that we did.
    • ice for 10 min
  8. spin 10 min at 10Krpm in SS34 tube
    • we spun using the table top centrifuge instead of the SS34 one at 4500 rpm for 2 min.
  9. collect supernatant in fresh SS34 tube (~10mL)
    • this was kinda weird. there were lipids and stuff sitting on the top surface and other gook sitting at the bottom. I had to get mostly everything in the middle. When I put the pipette tip in the top layer would stick to the tip, and everything that didn't stick would then mix back into solution. I got it though.
    • There was about 9mL of DNA after pipetting
  10. add 30mL EtOH
    • added more like 25-28mL because the EtOH can only hold about 40mL, so getting exactly 40mL in here was pushing it.
  11. precipitate ~20C for 60 min
    • we stopped here and will allow to sit for the weekend. To return on Monday...

Calculations

ODs

Apparently the number that the spectrometer provides can be interpreted ODs/mL.

Started with 0.958 in 10mL and need to make .0005OD in 25mL:

[math]\displaystyle{ \frac{.958}{.0005}= 1916 \qquad and \qquad \frac{25}{1916} = .013mL = 13uL }[/math]

That is how much of the starter culture Kelly was supposed to and did add to 25mL of YPD. Then we needed 10ODs of cells from the 25mL stocks so:

[math]\displaystyle{ \frac{10}{.892}=11.2mL \qquad \frac{10}{.802}=12.5mL }[/math]

The calculation that yielded 11.2 was for batch 1 and the other is for batch 2. It was at this point that we realized we would have enough extra to do two more batches hence, 1.1, 1.2, 2.1, 2.2

Spheroplasting

I don't remember much about the spheroplasting proportions. We made a 5mL solution and we needed to add 50mM of 2-ME. I think the stock was somewhere around 14M, but we ended up adding 87uL (turned out to be a 286x dilution), to go with the 150uL lyticase.

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