IGEM:Peking/2007/Switch: Plasmid mini prep: Difference between revisions
Chen Chongyi (talk | contribs) (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 (talk | contribs) |
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==Plasmid Mini Prep== | ==Plasmid Mini Prep== | ||
===procedure=== | ===procedure=== | ||
1. | 1.Pick single colonies，cultivate in 2mL LB media with some antibiotic to saturation. | ||
2. | 2.Turn on the centrifuge，set it to 1,3000rpm，4℃ | ||
3. | 3.Put 1.5mL culture into 1.5mL EP tube，centrifuge for 30s. | ||
4. | 4.Discard the culture, make the remaining pellet as dry as possible. | ||
5. | 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. | 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. | 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. | 8.Centrifuge for 4min. | ||
9. | 9.Move the supernatant to new tubes which have already been added 400uL phenol:choloform. Vortex and mix them gently. | ||
10. | 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. | 11.Centrifuge for 10min. | ||
If it is a low copy plasmid, quiescent for 30min before centrifuge. | |||
12. | 12.Pour out the supernatant after centrifugation. | ||
13. | 13.Put some 70% ethanol(stored in 4℃）into 1.5mL EP tube。Wash the pellet with 70% ethanol. | ||
14. | 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. | 15.Carry out the enzymatic digestion. The plasmid can be stored in 4℃. | ||
===Solution Components=== | |||
=== | |||
====Solution I==== | ====Solution I==== | ||
200mL | 200mL | ||
1M | 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 | ||
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 | ||
Constant-volumn to 200mL | |||
====Solution III==== | ====Solution III==== | ||
| Line 70: | Line 67: | ||
H2O 57mL | H2O 57mL | ||
==== | ====Phenol：Choloform==== | ||
100mL | 100mL | ||
Tirs saturated phenol（pH 8.0) 50mL | |||
Choloform 48mL | |||
isopentanol 2mL | |||
Quiescent overnight before any usage | |||
Revision as of 07:32, 18 October 2007
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
Tirs saturated phenol（pH 8.0) 50mL
Choloform 48mL
isopentanol 2mL
Quiescent overnight before any usage
