DNA isolation from yeast

互联网2013-09-05

563

<center> DNA isolation from yeast </center>

1) Start a 5 ml o/n culture
2) Pellet o/n culture in a purple screw cap tube (this may take several spins), and dump s/n.
3) Add 100 µl of STET to pellet and vortex briefly.
4) Add about a cap full of 0.45mm glass beads (make sure there are no beads in the cap threads and tighten well to prevent leakage).
5) Vortex for 5 min. (8 if using multi head vortex) at RT
6) Add another 100 µl of STET, and vortex briefly.
7) Place tubes in 100^o C heat block for 3 min. (cover tubes with weight). Alternatively, use boiling water bath for this step.
8) Cool briefly on ice (1-2min), and then use a blue tip to transfer as much liquid as possible to 1.5ml eppendorf tube (discard the glass beads).
9) Centrifuge at 4^o C for 10 min.
10) Transfer 100 µl of s/n to a fresh tube.
11) Add 500 µl of 7.5M ammonium acetate (stored at 4^o C) and mix briefly.
12) Incubate at -20^o C for 1 hr or overnight.
13) Centrifuge 10min. at 4^o C (should see a small white pellet).
14) Aliquot 200µl of ice cold 100% ethanol into to fresh eppendorf tubes. Transfer 100µl of s/n to these tubes.
15) Mix well and let stand at RT for about 5 min.
16) Centrifuge 10 min. at RT
17) Discard s/n, wash pellet with 200µl of 70% ethanol.
18) Centrifuge 5min., remove s/n, dry pellet in speed vac.
19) Re-suspend pellet in 20µl of TE or water, use 10µl to transform competent bacteria.

STET buffer:

• 8% sucrose in 50mM Tris pH 8.0
• 5% TritonX-100
• 50mM EDTA
• 7.5 Ammonium Acetate: 28.9g / 50mls