【共享】酵母电转改进PROTOCOL

小弟弟一次做电转，完全按照毕氏酵母说明书来，居然没长出来。第二次，按老板出国前留下的做，效果甚好！现贡献出来，望对大家有所帮助！

小弟刚做酵母表达，在DXY受益匪浅！看到不少关于酵母电转的PROTOCOL,但基本上是按INVITROGEN上的步骤来的。小弟第一次也是照本宣科，可是没出来，后来又按导师留给小弟的PROTOCOL做了一次，转化效果特好。先将其贡献给大家，希望对大家有所帮助：

Yeast Transformation Protocol (modified to a smaller volume)

Day 1

1.Inoculate 5mls YPD (in a 50 ml tube) with yeast strain (single colony on YPD plate or scraping from glycerol stock). Incubate O/N at 28-30 degrees, shaking.

Day 2

2.In early afternoon (3pm), inoculate 100mls of YPD (500ml flask) with 100ul of O/N culture (glycerol stock the rest).

Day 3

3.Spin cells down in 2 x 50ml tubes (1500g for 5 min at 4 degrees; JA10 rotor 2900rpm)
4.Resuspend pellets in 10mls Buffer A; incubate at 30 degrees for 15 mins (NO SHAKING, place tubes in 30 degree incubator). Fill up 50ml tube with ICE COLD sterile water.

Buffer A: 20mls YPD + 2ml 2M HEPES, pH 8 + 0.5ml 1M DTT.

5.Spin cells down again and resuspend in a total volume of 50ml ice cold water + 0.3ml 2M HEPES, pH8

6.Spin cells down again and resuspend (pool tubes) in 4 mls ice cold 1M sorbitol.

7.Spin cells for 5 min at 1500g, then resuspend in as low a volume as possible of ice cold 1M sorbitol (will be goopy)-try for less that 500ul.

8.Keep cells on ice and use same day.

Multicopy Transfection Selection (for higher expression)

(based on Bio Techniques (1999) 26:1042-4)

1. Mix 80-160ul of freshly prepared cells with 10-25ug of linearized DNA (DNA in 5-10ul of sterile water) and transfer them to an ice-cold (0 degrees) 0.2cm electroporation cuvette (BIO RAD genepulser).

2. Incubate the cuvette with the cells/DNA on ice for 5-10 minutes.

3. Pulse the cells according to the manufacturer’s instructions for yeast (Saccharomyces cerevisiae). 1.5kV, 25uF, 200 Ohm.

4. Immediately add 2ml of ice-cold 1M sorbitol with HEPES (10ml 1m Sorbitol + 100ul 2M HEPES pH8) to the cuvette. Transfer the cuvette contents to a sterile 15ml tube.

5. Let the tube incubate at 30 degrees without shaking for 1-2 hours.

后面就是涂板了，小弟就不啰嗦了！

至于加DTT的作用，很多地方又阐述，总之是可以提高转化效率，而HEPES的目的，我觉得可能是为维持pH值。上述只是小弟拙见，还望高手指点！
祝大家好运！