A.The PCR Reaction System

B.The double enzyme digestion system (Q.cut)

C.Ligation system

D.The SOE（Gene splicing by over lap extension） PCR

We use SOE to fuse different genes, which are difficult to cut or ligate. We achieve the fusing by over lap extension during PCR. First, we need to design 4 primers. Primer 1 & 4 are normal ones. The left part of primer 2 is normal primer, while the right part is primer for gene B. Primer 3 is just like primer 2. The right part of primer 3 is normal, while the left part is primer for gene A. Some base of primer 2 and primer 3 can reach complementary base pairing. Second, we amplify gene A by primer 1 & 2 and amplify gene B by primer 3 & 4 respectively. After that we can get extended gene A and gene B. Last but not least, we blend extended gene A & gene B can complemet each other and form the hybirdized strands. Under the function of DNA polymerase I, gene A and gene B can be the primer and templet of each other. After PCR, we can get fused Gene A-B.

https://2017.igem.org/File:Team-HUST-China-2017-SOE.jpg

E.Transformation into E. coli DH5α

The ligation product was transformed into E.coli DH5α strain.

If the vector was pET28a, the strain was grown in LB plate medium containing 10μg/ml kanamycin at 37°C.

If the vector was pBAD30, the strain was grown in LB plate medium containing 100μg/ml ampicillin at 37°C.

If the vector was pSB1C3, the strain was grown in LB plate medium containing 25μg/ml chloramphenicol at 37°C.