Cultivation of Bacteria
(a Extract the plasmid from the original bacteria
(b Transform the plasmid into the competent cell BL21 and Dh5α
(c Place in solid LB which contain kanamycin to culture the bacteria in 37°C constant temperature incubator for 12h
(d Pick the best monoclonal and put into liquid LB which contain kanamycin to amplify bacteria in 37°C constant temperature incubator for 12h
PCR
(a Add necessary buffers and mixs and primers to the template
(b Put the 20 μl tubes into the PCR machine
(c Set the temperature at 95°C for 30s, and then cycle the process of 95°C for 15s, 58.5°C for 15s, 72°C for 1min 35 cycles, and at last set 72°C for 5mins.
DNA electrophoresis
(a Make a 10% agarose gel by using 0.4g agarose and 40ml TAE with 100ml water
(b Add the products of PCR to the agarose gel
(c Move the agarose gel into the DNA electrophoresis device and run for 30mins
(d Move the agarose to under the UV light and photograph the result
SDS-Page
(a Add 0.5mM IPTG and 15 ml liquid LB and kanamycin to 15ml transformed BL21
(b Separate the 30ml solution into 10 test tubes equally, and cultivate separately in 16°C incubator for 6h, 12h, 18h, 24h, 30h and 36h
(c Extract the protein form the solution
(d Use ddH_2O, 30%Acr-Bis, separate the gel buffer, 10%APS, TEMED to make a piece of SDS-Page gel
(e Add the protein to the SDS-Page gel and add the Loading Buffer to the device
(f Open the power supply for 1h
(g Take out the SDS-Page gel and add the dye for 30mins
(h Wash the SDS-Page gel until can see the result
