IGEM:Peking/2007/Switch: Plasmid mini prep

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(New page: ==Plasmid Mini Prep== ===procedure=== 1.挑取单克隆菌落,在约2mL适当抗性的LB培养基中培养至饱和。 2.打开离心机,设定为1,3000rpm,4℃ 3.将约1.5mL培养...)
Current revision (09:34, 18 October 2007) (view source)
(Phenol:Choloform)
 
(One intermediate revision not shown.)
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==Plasmid Mini Prep==
==Plasmid Mini Prep==
===procedure===
===procedure===
-
1.挑取单克隆菌落,在约2mL适当抗性的LB培养基中培养至饱和。
+
1.Pick single colonies,cultivate in 2mL LB media with some antibiotic to saturation.
-
2.打开离心机,设定为1,3000rpm,4℃
+
2.Turn on the centrifuge,set it to 1,3000rpm,4℃
-
3.将约1.5mL培养物倒入1.5mL离心管中,盖管盖,适当标记,离心30s。剩余培养物保存在4℃。
+
3.Put 1.5mL culture into 1.5mL EP tube,centrifuge for 30s.
-
4.将培养液倒出,残存培养液用200uL枪头吸干净,使沉淀尽量干燥。
+
4.Discard the culture, make the remaining pellet as dry as possible.
-
5.将适量Solution I(保存于4℃)倒在1.5mL离心管中。每管细菌沉淀加入100uL分装后的 Solution I,盖管盖,用votex重悬至无可见的块状沉淀。
+
5.Put some Solution I(stored in 4℃)into 1.5mL EP tube. Every tube is added by 100uL Solution I,votex until there is no visible pellet。
-
6.将适量Solutin II(保存于室温)倒在1.5mL离心管中。开管盖,每管加入200uL分装后的Solution II,盖管盖,快速颠倒离心管5次,溶液变澄清,开管盖,同时可见透明丝状物。
+
6.Put some Solutin II(stored in room temperature)into 1.5mL EP tube. Every tube is added by 200uL Solution II,mix it by quick reversal for 5 times until the solution get clear. You can see some transparent thing like silk.
-
7.将适量Solution III(保存于4℃)倒在1.5mL离心管中。每管加入150uL分装后的Solution III,盖管盖,倒置离心管,温和震荡10次,可见白色粘稠不溶物。
+
7.Put some Solution III(stored in 4℃)into 1.5mL EP tube. Every tube is added by 150uL Solution III, put the tube upside-down and votex it gentlely for 10 times.
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8.离心4min。同时在新的1.5mL离心管上做好适当标记,每管加入400uL酚氯仿(保存于4℃,吸下层)。将各种溶液放回原处。
+
8.Centrifuge for 4min.
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9.离心后可见较紧致的白色沉淀。将上清用1000uL枪头吸入对应的加好酚:氯仿的离心管中,每个样品间换枪头。盖管盖,温和振荡混匀。
+
9.Move the supernatant to new tubes which have already been added 400uL phenol:choloform. Vortex and mix them gently.
-
10.离心2min,同时在新的1.5mL离心管上做好适当标记(此次为正式标记),每管加入800uL100%乙醇(保存于4℃)。
+
10.Centrifuge for 2min, move the upper layer to new tubes which have already been added 800uL 100% enthanol. Vortex gently.
-
离心后可见溶液分两层,两相间有少量白色沉淀。将上清用200uL枪头小心吸入对应的加好乙醇的离心管中(防止吸入白色沉淀),每个样品间换枪头。盖管盖,温和振荡混匀。
+
-
如果不慎将酚氯仿粘到手上,应尽快涂大量去垢剂后用水洗掉!
+
If got some phenol:choloform on the hand, immediately put some detergent on and wash with large quantity of water.
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11.离心10min,将溶液放回原处。
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11.Centrifuge for 10min.
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如是低拷贝质粒,应在离心前静置30min。
+
If it is a low copy plasmid, quiescent for 30min before centrifuge.
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12.离心后可见白色沉淀,倒出上清。
+
12.Pour out the supernatant after centrifugation.
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13.将适量70%乙醇(保存于4℃)倒在1.5mL离心管中。开管盖,每管加入200uL分装后的70%乙醇,盖管盖,清洗管壁和沉淀。
+
13.Put some 70% ethanol(stored in 4℃)into 1.5mL EP tube。Wash the pellet with 70% ethanol.
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14.离心1min,将乙醇倒出,残存的乙醇用200uL枪头吸干净,使沉淀尽量干燥。于37℃干燥10min至无明显的乙醇味道。同时溶解2×RNase。
+
14.Centrifuge for 1min,pour out the ethanol and make the pellet as dry as possible. Quiescent under 37℃ for 10min until there is no smell of ethanol.
-
每管加入20uL2×RNase,每个样品间换枪头,盖管盖,于37℃孵育。
+
Add 20uL 2×RNase into each tube and put it in 37℃.
-
15.配制适当的酶切体系对质粒进行检测。将质粒保存于4℃
+
15.Carry out the enzymatic digestion. The plasmid can be stored in 4℃.
-
将阳性质粒保存于-20℃,丢弃隐性质粒。如有需要,将阳性质粒对应的菌液送测序。丢弃剩余菌液。
+
===Solution Components===
-
 
+
-
===溶液配方===
+
====Solution I====
====Solution I====
200mL
200mL
-
1M 葡萄糖 10mL
+
1M Glucose 10mL
1M Tris•Cl (pH 8.0)                 5mL
1M Tris•Cl (pH 8.0)                 5mL
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0.5M EDTA (pH 8.0)         4mL
0.5M EDTA (pH 8.0)         4mL
-
定容至200mL,115℃ (6.895×104Pa)蒸汽灭菌15min。
+
Constant-volumn to 200mL,115℃ (6.895×104Pa) steam sterilize for 15min。
====Solution II====
====Solution II====
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10% SDS 20mL 1% SDS
10% SDS 20mL 1% SDS
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定容至200mL
+
Constant-volumn to 200mL
====Solution III====
====Solution III====
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H2O 57mL
H2O 57mL
-
====酚:氯仿====
+
====Phenol:Choloform====
100mL
100mL
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Tirs饱和酚(pH 8.0)(下层) 50mL
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Tris saturated phenol(pH 8.0) 50mL
-
氯仿 48mL
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Choloform 48mL
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异戊醇 2mL
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isopentanol 2mL
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静置过夜后使用。
+
Quiescent overnight before any usage

Current revision

Contents

Plasmid Mini Prep

procedure

1.Pick single colonies,cultivate in 2mL LB media with some antibiotic to saturation.

2.Turn on the centrifuge,set it to 1,3000rpm,4℃

3.Put 1.5mL culture into 1.5mL EP tube,centrifuge for 30s.

4.Discard the culture, make the remaining pellet as dry as possible.

5.Put some Solution I(stored in 4℃)into 1.5mL EP tube. Every tube is added by 100uL Solution I,votex until there is no visible pellet。

6.Put some Solutin II(stored in room temperature)into 1.5mL EP tube. Every tube is added by 200uL Solution II,mix it by quick reversal for 5 times until the solution get clear. You can see some transparent thing like silk.

7.Put some Solution III(stored in 4℃)into 1.5mL EP tube. Every tube is added by 150uL Solution III, put the tube upside-down and votex it gentlely for 10 times.

8.Centrifuge for 4min.

9.Move the supernatant to new tubes which have already been added 400uL phenol:choloform. Vortex and mix them gently.

10.Centrifuge for 2min, move the upper layer to new tubes which have already been added 800uL 100% enthanol. Vortex gently.

If got some phenol:choloform on the hand, immediately put some detergent on and wash with large quantity of water.

11.Centrifuge for 10min.

If it is a low copy plasmid, quiescent for 30min before centrifuge.

12.Pour out the supernatant after centrifugation.

13.Put some 70% ethanol(stored in 4℃)into 1.5mL EP tube。Wash the pellet with 70% ethanol.

14.Centrifuge for 1min,pour out the ethanol and make the pellet as dry as possible. Quiescent under 37℃ for 10min until there is no smell of ethanol.

Add 20uL 2×RNase into each tube and put it in 37℃.

15.Carry out the enzymatic digestion. The plasmid can be stored in 4℃.

Solution Components

Solution I

200mL

1M Glucose 10mL

1M Tris•Cl (pH 8.0) 5mL

0.5M EDTA (pH 8.0) 4mL

Constant-volumn to 200mL,115℃ (6.895×104Pa) steam sterilize for 15min。

Solution II

200mL

10M NaOH 4mL

10% SDS 20mL 1% SDS

Constant-volumn to 200mL

Solution III

200mL

5M KAC 120mL

HAC 23mL

H2O 57mL

Phenol:Choloform

100mL

Tris saturated phenol(pH 8.0) 50mL

Choloform 48mL

isopentanol 2mL

Quiescent overnight before any usage

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