IGEM:Peking/2007/Switch: Plasmid mini prep - Revision history

Chen Chongyi: /* Phenol：Choloform */

Chen Chongyi — Thu, 18 Oct 2007 14:34:43 GMT

Phenol：Choloform

←Older revision		Revision as of 14:34, 18 October 2007
Line 70:		Line 70:
	100mL		100mL

-	~~Tirs~~ saturated phenol（pH 8.0) 50mL	+	Tris saturated phenol（pH 8.0) 50mL

	Choloform 48mL		Choloform 48mL

Chen Chongyi: /* Plasmid Mini Prep */

Chen Chongyi — Thu, 18 Oct 2007 14:32:45 GMT

Plasmid Mini Prep

←Older revision		Revision as of 14:32, 18 October 2007
Line 1:		Line 1:
	==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.

-	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.

-	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.

-	11.~~离心10min，将溶液放回原处。~~	+	11.Centrifuge for 10min.

-	~~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.

-	12.~~离心后可见白色沉淀，倒出上清。~~	+	12.Pour out the supernatant after centrifugation.

-	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.

-	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
Line 50:		Line 47:
	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====
Line 59:		Line 56:
	10% SDS 20mL 1% SDS		10% SDS 20mL 1% SDS

-	~~定容至200mL~~	+	Constant-volumn to 200mL

	====Solution III====		====Solution III====
Line 70:		Line 67:
	H2O 57mL		H2O 57mL

-	====~~酚：氯仿~~====	+	====Phenol：Choloform====
	100mL		100mL

-	~~Tirs饱和酚（pH~~ 8.~~0）（下层）~~ 50mL	+	Tirs saturated phenol（pH 8.0) 50mL

-	氯仿 48mL	+	Choloform 48mL

-	~~异戊醇~~ 2mL	+	isopentanol 2mL

-	~~静置过夜后使用。~~	+	Quiescent overnight before any usage

Chen Chongyi: 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...

Chen Chongyi — Thu, 18 Oct 2007 13:51:56 GMT

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...

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℃。

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。

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。

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。

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。

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。

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。

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℃）。
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！

11.离心10min，将溶液放回原处。

如是低拷贝质粒，应在离心前静置30min。

12.离心后可见白色沉淀，倒出上清。

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。

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。

每管加入20uL2×RNase，每个样品间换枪头，盖管盖，于37℃孵育。

15.配制适当的酶切体系对质粒进行检测。将质粒保存于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。

===溶液配方===
====Solution I====
200mL

1M 葡萄糖 10mL

1M Tris•Cl (pH 8.0) 5mL

0.5M EDTA (pH 8.0) 4mL

定容至200mL，115℃ (6.895×104Pa)蒸汽灭菌15min。

====Solution II====
200mL

10M NaOH 4mL

10% SDS 20mL 1% SDS

定容至200mL

====Solution III====
200mL

5M KAC 120mL

HAC 23mL

H2O 57mL

====酚：氯仿====
100mL

Tirs饱和酚（pH 8.0）（下层） 50mL

氯仿 48mL

异戊醇 2mL

静置过夜后使用。