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).

THIS PAGE IS UNDER CONSTRUCTION

=Procedure=
 * 1) Grow yeast culture (I started with wt w303a)
 * 2) Inoculate started culture in liquid YPD.
 * 3) *Store in 30C for > 4hrs
 * 4) Measure OD 600nm
 * 5) Dilute to OD=0.0005 in 25mL YPD
 * 6) *Store in 30C overnight
 * 7) Measure OD (hopefully will be around .5)
 * 8) Spin 10 ODs of cells
 * 9) Resuspend pellet in 5mL of sheroplasting buffer (SB) with 50mM 2-ME and 150uL lyticase
 * 10) Incubate on ice
 * 11) *monitor OD of 1/4 dilutions in 1% SDS (let sit in SDS for 2 min before measuring)
 * 12) *OD should drop from .5 to < .1 over about 30 min
 * 13) Add 5mL Proteinase K Buffer (PK; to be made fresh)
 * 14) Incubate for 30 min at 65C
 * 15) Add 2mL 5M KOAc
 * 16) *ice for 10 min
 * 17) spin 10 min at 10Krpm in SS34 tube
 * 18) collect supernatant in fresh SS34 tube (~10mL)
 * 19) add 30mL EtOH
 * 20) precipitate ~20C for 60 min
 * 21) *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.
 * 22) spin 10 min
 * 23) resuspend pellets in 500uL TE
 * 24) *transfer to 2mL eppi
 * 25) add RNaseA to
 * 26) *incubate 37C for 30 min
 * 27) extract with Phenol/Chloroform
 * 28) EtOH precipitate DNA (~400uL plus 1.2mL EtOH)
 * 29) spin 10 min in microfuge
 * 30) dissolve DNA in 50-100uL TE
 * 31) measure DNA concentration on nanodrop

=Possible changes=
 * 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.