Fig. 4

An overview of the procedure for constructing the goal-insert, ordered cpDNA library. a Enzyme digestion prepares clonable cpDNA fragments of the desired size. The circular cpDNA within the unstained gel slices represents Wheat (Label 1), Maize (Label 2), Soybean (Label 3) and Barley (Label 4). The gel slices stained with ethidium bromide are Soybean circular cpDNA (label 5, 6), as the +ck of the circular cpDNA template in the slice. The UV then processes the gel slices into the image. b In-situ substitute for the desired size, cloneable DNA fragments. The enzyme-digested buffer has been substituted and concentrated by X solution using the MCE membrane as the substrate layer. The MCE membrane was dealt with cold radiation sterilization, and drip the 50 μL enzyme-digested reaction onto the MCE membrane. Using 0.6 mm spinners magnetic stirrer at 100 R/4 °C for 8 h, replace the enzyme-digested buffer with the X solution, and the X solution can be osmotically attained for 14–16 μL upon MEC membrane as the solvent of the clonable cpDNA fragment. c In-situ ligation for the good-quality vector/insert recombinant DNA. d Construction of the circular cpDNA library. e Verifying the cpDNA library by PCR. The colonies from the cpDNA libraries plate are verified by PCR. Black arrowheads show the target bands of the primer sets. The junction sites were validated by PCR, with one PCR primer on the vector and the other on the insert that complies with Table 4. The DL2000 Marker is placed on the far left