IGEM:Peking/2007/Switch: Plasmid mini prep

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