From 2013.igem.org
Plasmid Purification
QIAprep Spin Miniprep Kit (QIAGEN)
Contents ：
Buffer P1
Buffer P2
Buffer PB
Buffer EB
Collection tubes
Eppendorf Tubes
Vortex
Centrifugal machine
Collection tubes
Eppendorf Tubes
Vortex
Centrifugal machine
Add 2 ml of culture medium of E.coli which cultured in overnights into an Eppendorf
tube. Then, centrifuge the tube in room temperature (23~25℃), 130000 rpm for one minute.
Add 250 μl Buffer P1 into the tube. Then, stir it with Vortex until deposition disappears.
Add 250 μl Buffer P2 and mix the contents by inverting the tube (Don’t use the Vortex)
Add 350 μl Buffer N3 and mix the contents by inverting the tube (Don’t use the Vortex.
Centrifuge the tube in room temperature, 13000 rpm for 10 minutes.
Move the supernatant by a pipette from the tube to the QIAprep Spin Column.
Centrifuge in room temperature, 13000 rpm for 1 minute and throw away the flow through liquid which collected at the bottom of the column.
Add 500 μl Buffer PB and centrifuge the tube in room temperature, 13000 rpm for 1 minute. Then, throw away the flow through liquid.
Add 500 μl Buffer PE and centrifuge the tube in room temperature, 13000 rpm for 1 minute. Then, throw away the flow through liquid.
Set the upper part of the QIAprep Spin Column into another Eppendorf tube.
Add 50 μl EB buffer and left it at room temperature for 1 minute.
Centrifuge in room temperature, 13000 rpm for 1 minute. The flow through liquid is the plasmid DNA solution.
Restriction Enzyme Digestion
Reagents and Materials
Eppendorf tube
milliQ
DNA solution
Digest Buffer
Restriction enzyme
Mix these solutions with the following quantities.
Pattern1
| milliQ | 12 µl |
| DNA solution | 5 µl |
| Difest Buffer | 2 µl |
| Restriction enzyme | 1 µl |
| total | 20 µl |
|
Pattern 2
| milliQ | 12 µl |
| DNA solution | 5 µl |
| Difest Buffer | 2 µl |
| Restriction enzyme ① | 1 µl |
| Restriction enzyme ② | 1 µl |
| total | 20 µl |
|
- Incubate them at 37℃ for 1 hour.
- Incubate them at 60℃ for 15 minutes.
Electrophoresis
Reagents and Materials
- Agarose gel
- TAE Buffer
- Loading Buffer
- DNA solution
- Eppendorf tube
- Ethidium bromide
- Set gel in the electrophoresis tank anf pour the TAE Buffer into the tank.
- Put DNAsolution and Loading Buffer in the ratio of 1:2 into the Eppendorf tube and mix them.
- Load samples into wells.
- Do Electrophoresis at 100V for 40min.
- After electrophoresis, soak the gel into water including ethidium bromide for 20min.
- Observe the bands by ultraviolet radiation.
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