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YAC DNA preparation

YACs can be grown with single or double selection. With a single selection marker (-Ura), the YACs grow somewhat faster. With double selection (-Ura, -Trp) you get a better yield of some YACs. The isolation procedure is the same. Use 1.5X medium for growth and 1X medium + 2% agar for plates.

  1. Grow colonies on a plate at 30°C for ~48 Hrs. Pick a colony into 10ml of medium in a 50ml tube. Grow for ~48 Hrs, until saturated. The culture should be pink if using both selection markers.
  2. Add all 10 ml to 400 ml medium in a 2L flask.
  3. Grow for ~30 Hrs, until saturated.
  4. Centrifuge 10 min, 5000 rpm.
  5. Aspirate to ~50 ml, transfer to polypropylene (orange cap) tube.
  6. Spin 5 min at 7000 rpm and aspirate.
  7. Wash with 2 x 50 ml ice cold 50 mM EDTA. Resuspend in 8 ml 50 mM EDTA, warm to RT.
  8. Add 5 ml Solution I.
  9. Add 20 ml 1% low melt agarose in 100 mM EDTA at 42°C. Quickly vortex and pour into a box (10 x 6 cm), let set at RT.
  10. Add 20 ml solution II. Seal with parafilm. Put at 37°C overnight. Seal really well so BME doesn't escape.
  11. Cool to RT. Take off solution.
  12. Add 20 ml solution III. Seal well again and put at 50°C overnight.
  13. Cool to RT. Take off solution.
  14. Dialyze against 4 changes of T10E50 pH 8.0 at 4°C.
  15. Store in T10E50 pH8.0 at 4°C.

Solution I
5 ml SCE
7.5 mg zymolase (=lyticase)
250 µl BME

Solution II
20ml 0.5 M EDTA, pH 9.0
1.5ml BME

Solution III
20ml 0.5M EDTA, pH 9.0
670µl 1% Sarcosyl (N-lauryl sarcosine)
20mg proteinase K.

9.11 g Sorbitol = 1M
5ml 1M Na Citrate pH 5.8
1ml 0.5 M EDTA pH 9.0
44ml H2O