User:Anthony Salvagno/Notebook/Research/Yeast Genomic DNA Prep
This procedure should describe all/most of the steps involved in extracting yeast genomic DNA. Protocols for creating certain chemical solutions can be found in my Notebook on the days I created them (see Table of Contents on the main Research page).
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- Grow yeast culture (I started with wt w303a)
- Inoculate started culture in liquid YPD.
- Store in 30C for > 4hrs
- Measure OD 600nm
- Dilute to OD=0.0005 in 25mL YPD
- Store in 30C overnight
- Measure OD (hopefully will be around .5)
- Spin 10 ODs of cells
- Resuspend pellet in 5mL of sheroplasting buffer (SB) with 50mM 2-ME and 150uL lyticase
- Incubate on ice
- 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
- Add 5mL Proteinase K Buffer (PK; to be made fresh)
- Incubate for 30 min at 65C
- Add 2mL 5M KOAc
- ice for 10 min
- spin 10 min at 10Krpm in SS34 tube
- collect supernatant in fresh SS34 tube (~10mL)
- add 30mL EtOH
- precipitate ~20C for 60 min
- this is a stable state, so it could be a good stopping point. You would be able to end here for the day and go home.
- spin 10 min
- resuspend pellets in 500uL TE
- transfer to 2mL eppi
- add RNaseA to
- incubate 37C for 30 min
- extract with Phenol/Chloroform
- EtOH precipitate DNA (~400uL plus 1.2mL EtOH)
- spin 10 min in microfuge
- dissolve DNA in 50-100uL TE
- measure DNA concentration on nanodrop
- In step 7 (adding lytocase), in my first trial we needed to add an extra 100uL of lyticase because the reaction was taking too long. This worked well.
- Total reaction time took 105 min after adding more lyticase at the 60 min point.