Shortcut Yeast transformation

Friday, October 24, 2003

Description
Method to transform plasmid DNA into yeast using a small amount of at-the-bench time. Requires 1 overnight to complete.

Procedure
1. Take fresh overnight of yeast cells from liquid or media rich plate.

2. Pellet cells, wash with ddH2O, and resuspend in 1xLiCl/ 40% PEG solution. Use (200 ul) x (number of transformation) for resuspension volume.

3. Aliquot 200ul of cells into a microfuge tube for each transformation. Include 5ul of miniprep DNA (~500 ng) + 8ul of herring sperm DNA (carrier DNA). Add 300ul 1x LiCl/ 40% PEG. Invert to mix.

4. Leave overnight on bench.

5. Heat shock 6 minutes at 42 degrees Celsius.

6. Spin 15 seconds at 13,200 rpm (~16,000g). Pour off PEG.

7. Wash with 1ml ddH2O. Use a toothpick to resuspend the cells (spin the toothpick with your fingers like a propeller). Spin at 8000 rpm for 15 seconds and pour off the H2O.

8. Resuspend the cells in 300-400ul of ddH2O.

9. Plate 10% and 50% for single transformation. Plate at least 50% for double transformation.

Recipes

Supplies

Tips

(责任编辑：泉水)

搜索

热门标签:

Shortcut Yeast transformation