Library construction and single-clone sequencing

Library construction and single-clone sequencing

Plasmid linearization

1, Prepare the enzyme digestion mixture: 

2, Incubate the mixture at 37°C for 2 hours.

3, Take 1 μL of the mixture to run agarose gel electrophoresis. The undigested plasmid was loaded as a negative control. Make sure that the plasmid was thoroughly digested.

4, Purify the linearized plasmid by DNA purification kit (Axygen, AP-PCR-250G). 

5, Measure the concentration of purified plasmid using a nucleic acid quantification machine (Such as Allsheng, nano-200, or other equivalents).

6, Store the digested plasmid in -20°C.

CDS amplification

1, Design the primers for Gibson assembly manually or via Graphpad software. The CDS fragment amplified by this pair of primers should contain the initiation codon and exclude the stop codon. The ligated wild-type CDS sequence should be in frame of the 6X-myc sequence in the pCS2-MT plasmid.

2, Amplify the CDS from the cDNA using high fidelity DNA polymerase (Vazyme, P505-d1). The PCR mixture was formulated as follow:

The PCR procedure was:

3, Check the PCR products by agarose gel electrophoresis.

4, Purify the PCR products by DNA purification kit (Axygen, AP-PCR-250G).

5, Measure the concentration of purified PCR products using a nucleic acid quantification machine (Allsheng, nano-200).

Gibson assembly

1, Combine the CDS fragment with the linearized PCS2-MT using the Gibson assembly kit (Yeason, 10911ES20) following the instruction:

2, Make the mixture as follow:

3, Incubate at 50°C for 20 min.

4, Put it on ice for 5 min.

Transform the DNA into the bacteria.

1, Take 100 μL DH5α competent cell from the -80°C refrigerator and thaw on ice.

2, Add 10 μL ligation mixture and mix them gently.

3, Incubate for 30 min on ice.

4, Heat-shock the competent cell at 42°C for 60 s.

5, Put it on ice immediately and incubate for 2 min.

6, Spread it on LB solid medium containing 100 μg/mL ampicillin.

7, Incubate at 37°C for 14 h.

Colony PCR

1, Pick up a certain number of colonies as you want. Mix each colony with 10 μL of ddH₂O in separate tubes, and that will serve as the DNA template in the following colony PCR.

2, Make colony PCR mixture:

3, The PCR procedure:

4, Run colony PCR products on agarose gel electrophoresis,

5, Pick up colonies that have positive bands and expand them.

Bacteria expanded culture

1, Add the remaining bacteria to 1.8 mL LB liquid medium containing 100 μg/mL ampicillin in 24-well cell culture plates.

2, The bacteria medium was cultured at 37°C for 14 h. The rotational speed of the shaker was 200 r/min.

Sanger sequencing

The bacteria culture of each colony was subjected to Sanger sequencing. The sequencing company will extract plasmids separately and apply them to Sanger sequencing.