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大肠杆菌DH5alpha感受态细胞制备
Author: 戴沙, updated date: , view: 5, Q&A: 0

Materials and Reagents

所有称量过程需要尽量精确!!!

A. PIPES: 0.5M(PH6.7)  

 15.1 g PIPES1,4-哌嗪二乙磺酸)溶于80 ml ddH2O ,利用5M KOH或者NaOHPH6.7,最后定容100 ml,过滤灭菌,存于-20℃

BInoue buffer 配方(1L

Reagent

Amount per   liter

Final Concentration.

MnCl2.H2O

10.88g

55 mM

CaCl2.H2O

2.20g

15 mM

KCl

18.65g

250 mM

PIPES0.5M(PH6.7)

20 ml

10 mM

ddH2O

to 1 L


过滤灭菌后保存于-20℃,分成小份备用。

C.灭菌。所有需要用到的锥形瓶,离心管、配装试剂的容器均需利用双蒸水冲洗多次,并灭菌。


Procedure

所有转接菌、悬菌操作都需要在擦净灭菌的超净工作台进行!!!

1. -80℃拿出Ecoli.原液,再LB板子上划线,37℃12-16 hours)。(早上划线活化)

2. 挑单菌落转接入25ml LB锥形瓶中,置于37℃ 200rpm摇床上,培养6-8 hours。(晚上摇单菌落)

3. 将摇过夜的菌种按照1:15的量接种至装有500ml LB 2L锥形瓶中,置于18-22℃摇床中培养,每隔一小时测一次OD600值,待其OD6000.45-0.5时进行下一步操作。

4. 将菌液OD6000.45-0.5)置于冰上冰浴10 min,离心机遇冷至4℃Inoue buffer在冰上预冷待用。

5. 收集菌体,4℃ 2000g离心10 min

6. 尽量将上清倒干净(倒去上清后可在灭菌的吸水纸上沥干,超过1-2 min)。

7. 在冰上按照每250 ml菌液用80 ml 0.5M (PH6.7) Inoue buffer 悬浮菌体洗去菌体上的LB500ml菌液用160 ml Inoue buffer悬浮菌体)。Gently

8. 将重悬的菌液2000g 4℃离心10 min

9. 再按照每80 ml菌液用20 ml 0.5M (PH6.7) Inoue buffer 悬浮菌体(160 ml菌液用40 ml Inoue buffer悬浮菌体)。Gently

10. 在冰上按照每20 ml菌液加入1.5 ml DMSO40 ml菌液用3 ml DMSO)。一定要按量加入DMSO(少则达不到保护剂效果,多则毒害细胞),同时摇匀。Gently

注意:DMSO切不可放于-20℃或更低,避免爆炸!!!

DMSO不可暴露光下,避免氧化!!!

11. 加入DMSO混匀后置于冰上冰浴10 min-20℃预冷1.5 ml离心管。

12. 在冰上按照50 ul或者100 ul分装,并立即用液氮速冻,后放于-80℃保存备用。


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