A: Preparation of Vector
(1)Enzyme digestion: digest plasmid vector with restriction enzyme(s) to generate the linearized vector. Purify the digested vector using Gel Extraction Kit (Cat. No. EG101).
(2)PCR amplification: prepare the linearized vector by high-fidelity DNA polymerase. If a single expected band is generated, use PCR Purification Kit (Cat. No. EP101) to purify the product. Otherwise, use Gel Extraction Kit to recover the product.
In order to increase the positive cloning efficiency, we suggest using DMT enzyme to digest plasmid template before PCR purification or gel extraction. Add DMT enzyme (Cat. No. GD111) after PCR amplification (1 μl of DMT enzyme for a 50 μl PCR system), and incubate at 37°C for 30 minutes.
B: Preparation of Inserts
(1)Forward primer (5’-3’): 15-25 nt homology of linearized vector + 20-25 nt target specific sequence. Reverse primer (5’-3’): 15-25 nt homology of linearized vector + 20-25 nt target specific sequence.
(2) Primers for multiple fragments
(3)We suggest to use high-fidelity DNA polymerases to generate both the linear vector and fragments.
•Use 0.2-0.4 μM ( final concentration) primers for PCR.
•Use 60-68°C as annealing temperature.
(5) Purification of target DNA fragments
•To increase the cloning efficiency, if the recombinant vector has the same selection marker as the parental plasmid for PCR fragments, pretreat the PCR fragments with DMT enzyme before purification.
•If product is single band, we recommend using PCR Purification Kit (Cat. No. EP101) to purify your fragments.
•If products are multibands, we recommend using Gel Extraction Kit (Cat. No. EG101) to recover your fragments.