酵母細(xì)胞的細(xì)胞壁比較厚，不容易破壁，不如大腸桿菌的質(zhì)粒 容易提取，最近做了些釀酒酵母的實(shí)驗(yàn)，從釀酒酵母中提取質(zhì)粒，現(xiàn)在就總結(jié)下實(shí)驗(yàn)的方法和步驟。

酵母細(xì)胞質(zhì)粒提取 步驟

1. 接種單菌落(待檢測(cè)酵母細(xì)胞)于25mLYNB(補(bǔ)加氨基酸 營養(yǎng)物)培養(yǎng)基中，30℃振蕩培養(yǎng)過夜。

2.第二天取一滴菌液于進(jìn)行顯微鏡下觀察,目鏡用16,物鏡用40倍觀察細(xì)胞壁破碎前的狀態(tài),其成桿狀,流動(dòng)性比較下.

3.取10ml的培養(yǎng)酵母菌液,5000g離心3min,棄上清液.加入5ml的1倍TE懸浮.

4.將懸浮液倒入高壓破壁儀的樣品管中,利用高壓破碎機(jī)進(jìn)行破碎細(xì)胞壁,壓力加到20Mpa,停留15s,降壓,反復(fù)來回壓3次.取出細(xì)胞液,取一滴于顯微鏡下觀察,如果細(xì)胞呈不規(guī)則的球狀時(shí),而且其流動(dòng)性 比較大,說明其細(xì)胞壁已經(jīng)破碎成為原生質(zhì)體.

5.取2個(gè)EP管,每管加入1.5ml上述的細(xì)胞液,12000g離心5min,收集原生質(zhì)體.棄取上清液,每管加入300ul10%的SDS溶液,混勻冰浴5min,進(jìn)行破原生質(zhì)體.

6.然后加入150ul的tris飽和酚,和150ul的鹵仿異戊醇混合液(鹵仿:異戊醇=24:1),混勻,12000g,離心10min.

7.將水相移到另一EP管中,加入等體積的鹵仿異戊醇混合液(鹵仿:異戊醇=24:1), 混勻,12000g,離心10min.

8. 將水相移到另一EP管中,加入1/10體積的3M KAC溶液和2倍體積的無水乙醇,放入-20℃冰箱中

9.1h,12000g離心10min,倒出乙醇,等干燥后加入1ml的70%的無水乙醇,混勻,12000g,離心10min.

10.棄去乙醇,等室溫干燥后,每管加入20ul的TE溶液(如要去處RNA酶,加入1ul的100mg/ml濃度的RNA酶),放入-20℃冰箱即可.

11.跑電泳進(jìn)行檢測(cè)是否從酵母菌中提出質(zhì)粒了.

酵母破壁方法

我給你介紹兩個(gè)必叫簡(jiǎn)單點(diǎn)的酵母破壁方法，非常實(shí)用，我作過很多實(shí)驗(yàn)，這兩個(gè)方法是我自己總結(jié)出來的，希望對(duì)你有用。

酵母的細(xì)胞壁比較厚,不易破，而且細(xì)胞壁中含有的一些成分容易影響以后的實(shí)驗(yàn)，所以破壁的步驟非常關(guān)鍵!其他步驟只要你按照說明就可以了。

1. 酚氯仿劇烈振蕩方法破壁：培養(yǎng)好的1ml酵母細(xì)胞在STE溶液中洗滌兩次后，用70ulTE緩沖液重旋!加入50ul玻璃珠(sigma公司)，加入80-100ul酚氯仿，劇烈振蕩5-10分種后，使用氯仿抽取去除酚和蛋白，然后按照其他步驟沉淀就可以得到你的質(zhì)粒，DNA的提取也可以使用這種方法。

2.反復(fù)凍融破壁：培養(yǎng)好的1ml酵母細(xì)胞在STE溶液中洗滌兩次后，加入200ul緩沖液[2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)]，使用液氮和96-98度沸水反復(fù)凍融3-5次，然后使用酚氯仿抽提蛋白!其余步驟參考其他文獻(xiàn)

3.下面兩個(gè)文獻(xiàn)對(duì)你會(huì)有所幫助!請(qǐng)參考!不錯(cuò)的方法!

For yeast plasmid extraction we use the following method:

Buffer A: 100 mM NaCl 10 mM Tris-HCl (pH 8) 1 mM EDTA 0.1 % SDS

1. Culture the plasmid-harboring cells overnight in 1 - 2 ml of medium. Cells with 1 - 4 OD600/ml are needed.2. Pulse 5-10 sec at 15'000 rpm (maximum speed) (in an Eppendorf tube).3. Throw away the supernatant.4. Resuspend in 200 µl of buffer A, keep cells on ice.5. Add glass beads just before the solution surface, mix with a vortex during 1 minute and sonicate.6. Add 200 µl of phenol.7. Vortex another minute.8. Centrifuge at 15'000 rpm for 1 minute.9. Throw away the phenol phase.10. Add 200 µl of phenol and reextract the same way.11. Take the water solution (about 200 µl) which contains the plasmids and treat with Glassmilk: add 600 µl of NaI solution and 5 µl Glassmilk suspension, put the tubes on the wheel for 5 min., then pellet the Glassmilk/DNA complex (pulse 5 sec), remove the supernatant and set aside, then wash the pellet 3 times with 300 µl New Wash, then pulse for 5 sec to remove the New Wash.12. Elute the DNA into water (20 µl) or TE buffer.

When not using the GeneClean-kit, another protocol can be applied:Steps 1 to 9 remain the same as above. Then:

10. Add 200 ml phenol/chloroform 1:1 and vortex.11. Centrifuge at 15'000 rpm for 1 minute.12. Throw away the phenol phase.13. Add others 200 µl of chloroform and reextract the same way.14. Take the water phase and adjust the volume to 400 ml with water.15. Add 40 ml 3M NaCl.16. Add ca. 1 ml of ethanol 100 %.17. Put the tube at -20 0C for about 10 min..18. Centrifuge at 4 0C for 10 min. at maximum speed.19. Throw away the supernatant.20. Wash the pellet once with ethanol 80 % and once with ethanol 100 %.21. Dry the pellet by putting the tube upside down on a Kleenex.22. Resuspend the pellet in 50 ml TE buffer.

酵母質(zhì)粒提取可以用試劑盒提取，也可以直接用裂壁酶處理后采用堿裂解法提取質(zhì)粒，缺點(diǎn)就是提取的量很少，還會(huì)容易出現(xiàn)假陽性現(xiàn)象，明明有卻條帶，檢測(cè)卻不正確。