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Protocol

PCR

1.Add 100μL of reagent solution(Table) to microtube.
2.Dispense 20μL PCR solution to PCR tube.
3.Amplifty target DNA with PCR program.
4.Confirm the band of DNA by agar gel electrophoresis.

reagent name	Volume(μL)
TaKaRa Ex Taq	0.5
10×Ex Taw buffer	10
dNDP Mixture	8
template DNA	2
fowerd primer	4
reverse primer	4
sterile water	71.5
total	100

 

Transformation
　Preparation of E. coli contain particular plasmid

1.Incubate frozen competent cell on the ice.
2.Add 2μL of plasmid to competent cell.
3.Incubate for 15 minutes on the ice.
4.Incubate for 45 seconds at 42°C.
5.Incubate for 2 minutes on the ice.
6.Add 250μL LB medium and cultivate for 1 hour at 37°C.
7.Spread culture medium on LB agar plate with appropriate antibiotic.
8.Incubate over night at 37°C.

 

Restriction enzyme digestion of DNA

1.Mix DNA and restriction enzyme(Table).
2.Incubate over night at 37°C.
3.Confirm the band of DNA by agar gel electrophoresis.

reagent name	Volume(μL)
DNA	15
EcoR I	1
Xba I	1
buffer M	2
steril water	1
total	20

 

Plasmid extraction
　Preparation of plasmid extracted from E. coli

1. Pick up single colony from agar plate and cultivate it in 20 mL LB medium containing   
  appropriate antibiotic overnight at 37°C.
2. Move the culture medium to 50 mL falcon.
3. Centrifuge for 5 minutes at 6000rpm and 4 °C and discard solution.
4. Add 2 mL Solution1, the pellet.
5. Add 4 mL Solution 2, invert tube and stored 3 minutes on ice.
6. Add 3 mL Solution 3, invert tube and stored 5 minutes on ice.
7. Centrifuge for 10 minutes at 9,500rpm and 4 °C.
10.Take aqueous layer to new 50 mL falcon.
11.Add 40 μL RNase, invert tube and incubate for 30 minutes.
12.Add 2 mL phenol chloroform mixture.
13.Centrifuge for 5 minutes at 9,500rpm and 4 °C. 
14.Take supernatant to new 50 mL falcon.
15.Add 3 mL chloroform solution and misce.
16.Centrifuge for 3 minutes at 9,500rpm and 4 °C.
17.Take 3 mL supernatant to new 50 mL falcon.
18.Add 300 μL sodium acetate.
19.Add 7.5 mL 100%ethanol.
20.Centrifuge for 20 minutes at 9,500rpm and 4 °C and discard solution.
21.Add 8 mL 70% ethanol.
22.Centrifuge for 20 minutes at 9,500rpm and 4 °C and discard solution.
23.Add 50 μL TE buffer and tapping.
24.Preserve at freezer.