Protocols

PCR amplification fragments

1. PCR reaction system

Place in a PCR amplifier (Adjust the time according to the instructions).      

Agarose gel preparation for electrophoresis

1. Prepare with 1% agarose gelatin.
2. Weigh 0.5 g of agarose into a Conical flask and add 50 ml of Tris-acetate EDTA(TAE).    
3. Heat in the microwave until transparent.
4. Select the comb according to the fragment length and insert a comb at the top of the mold to form the sample well.    
5. After the gel is polymerized, the comb can be removed.
6. Place gel in the electrophoresis chamber. Gels should be completely submerged in buffer to allow ion flow and prevent gel drying.

Loading buffer

1. Load the marker into the first well using a micropipette and allow all the samples to “fall” into each of the following wells
2. Fill the upper and lower chambers with running buffer and apply an electric potential on the gel at a constant voltage of 120 V for 30min.
3. Remove the gel from the gel tray and expose the gel to UV light. DNA bands show up as fluorescent bands.

Recycling of DNA from Agarose Gels

1. Excise a gel slice containing the DNA of interest.
2. Place the gel slice containing the DNA sample into an EP tube. Add 30μl of XP2 Binding Buffer.
3. Melt the gel slice at 70°C for 15 minutes. Make sure that the gel slice is melted.
4. Pour in the Spin column to collect Tube and centrifugal (12000rpm 1min).
5. Discard the buffer.
6. Add 700μl SPW Buffer to elute 2 times. Centrifuge the solution (12000rpm 2min).
7. Replace the lower part with an EP tube.
8. Add 30μl Elution buffer and heat for 2 min at 60°C. Centrifuge the solution (12000rpm 1min).
9. Nucleic acid quantification.

Culture monoclonal colonies

1. Add 900 μl of antibiotic-free LB liquid medium and incubate at 180 rpm shaking for 45 min.    
2. Centrifuge at 6000 rpm for 5 min.
3. Inoculate onto Luria-Bertani (LB) medium containing Kanamycin. Incubate the culture overnight at 37°C.

Isolate and purify plasmids

1. Add 200 µl of Alkaline lysis solution II to each bacterial suspension.
2. Add 150 µl of ice-cold Alkaline lysis solution III.
3. Centrifuge the bacterial lysate at 12000 rmp for 5 min at 4°C in microfuge. Transfer the supernatant to a fresh tube.  
4. Add an equal volume of phenol: chloroform.
5. Adding ethanol into the supernatant. Mix the solution by vortexing.
6. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 min at 4°C in a microfuge.

Plasmid restriction digestion

1. Restriction digestion system

The centrifuge tube was placed in a 37 ℃ water bath overnight