KOF DNA Polymerase
This enzyme is a genetically modified high-fidelity DNA polymerase with extremely strong 3 '→ 5' exonuclease activity (Proof reading activity). When base mismatch occurs in the polymerization reaction, the correction activity of the polymerase can remove the mismatched base. Compared with ordinary pfu, it has higher fidelity, about 50 times that of ordinary taq and 6 times that of ordinary pfu. Its amplification performance is also higher than that of ordinary Pfu, successfully overcoming the defect of low amplification efficiency of ordinary PFU enzyme.
Product composition:
Component JN0017-250U
KOF DNA polymerase (1U/ul) 250ul
DNTP mixture 250ul
10X KOF Buffer with Mg2+2ml
Product Usage: Used for PCR reactions that require high fidelity, including cloning PCR, DNA fragment splicing, introducing mutations, whole gene synthesis, and DNA fragment complementation.
Usage suggestions:
The PCR product produced by KOF DNA Polymerase has a smooth end and no 3 'end "A" protrusion. There are several cloning schemes for its PCR product.
1. Before PCR, the primer was modified with 5 'phosphorus or the PCR product was phosphorylated before being directly cloned into a smooth terminal vector (see experimental protocol).
2. Add A to the 3 'end of the product before connecting it to the T carrier (see experimental protocol).
3. Introduce a 15bp homologous sequence with the vector into the primer, and use BalbRec PCR product one-step directional seamless cloning kit (Cat # JN0001) to directly connect to the target vector.
Due to the corrected activity of KOF DNA polymerase, the primer can be partially degraded from the 3 'end. Therefore, when designing primers, the length of the primers should be appropriately increased, with an ideal primer length of 20-30bp. In addition, in order to reduce primer degradation caused by 3 '→ 5' exonuclease activity, a reaction system should be prepared on ice as much as possible, and finally KOF enzyme should be added.
Application examples:
JN0017
Legend 1) In a 50ul amplification system, 5ng λ Amplification results of 0.6kb-12.0kb fragments using DNA as a template.
Swim lane 1: 0.6kb; Swim lane 2: 1.0kb; Swim lane 3: 2.0kb; Lane 4: 3.0kb; Lane 5: 4.0kb; Lane 6: 8.0kb; Lane 7: 10.0kb; Lane 8: 12.0kb; Swimming lane M: 1kb DNA Marker;
JN0017
Legend 2: Sensitivity amplification results using 50ng~0.05ng plasmids as templates in a 50ul amplification system.
Swim lane 1:50ng; Swim lane 2:5ng; Lane 3: 0.5ng; Swim lane 4: 0.05ng; Swim lane M: DNA 2000 ladder
Common reaction systems (50 μ L)
10X KOF Buffer 5 μ L
Upstream primers 0.2-1.0 μ M (final concentration)
Downstream primers 0.2-1.0 μ M (final concentration)
DNT
