Plant Selection Plates

 

Making the plates:

1) Dissolve 4.3 grams of MS salts per liter of H2O.

2) pH to 6.8 with KOH.

3) Add 8 grams Difco Bacto Agar per liter.

4) Autoclave 20 min, slow exhaust.

5) Cool to 50 C (approximately, by checking with hand), add the appropriate antibiotic. Usually Kanamycin 50 ug/ml or Hygromycin 50 ug/ml.

6) Pour into small plates in sterile environment, let cool > 1 hr.

7) One often needs to dry the plates in a laminar flow hood for 20-40 min before use.

8) Use sterile toothpick and sterile 3MM papers to transfer seeds to plates, if plating known transgenic plants at low density.

Selection of transformants at high density.

Materials

-Bleach solution.

-Sterile autoclaved water

-Sterile autoclaved 0.1% agarose, made the day before so it becomes viscous.

-MS Kan plates

All work done in sterile hood! Best to move microfuge and vortexer near hood.

1. Make 100 mls 50% bleach, 0.1% tween 20. 50 mls bleach plus 100ul tween.

2. Aliquat seeds in 1.5ml eppindorf tubes 100ul each tube (each tube is for one small petri plate).

3. Add 1ml of bleach+tween to each tube, vortex very lightly, let sit 5 min (not more or they die).

4. Rinse seeds with sterile water 4-5 times.

-Spin 10 seconds in microfuge at 3000 RPM to sediment seeds.

-Remove as much liquid as possible with 1ml pipetman with blue tip.

-Add 1ml sterile water and repeat.

5. After last wash, resuspend in 1ml sterile 0.1% Agarose, vortex very lightly, drip onto small plates in small drops with 1ml pipetman.

6. Dry with lids off in hood for 30 minutes, or until not dripping. Wrap plates with porous tape (from Carolina Biological Supply).

7. Stratify seeds as always in cold room for 3-7 days.

8. Grow under fluorescent lights on white styrofoam lids on the bottom shelf to prevent condensation. Usually can score transformants at 10 days. Transformants make true leaves and good roots and stay green.