User:Jarle Pahr/Enzymes

Notes on various enzymes:

Polymerases

(Polymerase not mainly used in PCR. For polymerases used in PCR, see PCR page):

T4 DNA polymerase: Posesses 5' -> 3 DNA polymerase and 3' -> 5' exonuclease activity. Used in SLIC. Does not show strand displacement activity. T4 DNA polymerase is active in NEBuffers 1-4 and NEB T4 DNA ligase buffer when supplemented with BSA. T4 DNA polymerase can be inhibited by adding EDTA.

T5 Exonuclease: A 5' -'3 exonuclease. Used in Gibson Assembly.

Klenow fragment: Fragment of E. coli DNA polymerase I, retaining 5'-3' polymerase activity and 3’ → 5’ exonuclease activity, but without 5' → 3' exonuclease activity. The 3'->5' exonuclease activity of the Klenow fragment is roughly 200 times less than that of T4 DNA polymerase (http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/t4dnap.html). Klenow fragment is commonly used to generate blunt ends by overhang fill-in. Klenow fragment is active in NEBuffers 1-4 and NEB T4 DNA ligase buffer. Klenow fragment can be heat-inactivated by incubation at 75C for 20 minutes.

T7 Exonuclease: 5'->3' exonuclease. Active in NEBuffer 4.

Lambda exonuclease: 5'->3' exonuclease. Unable to initiate digestion at nicks or gaps. Used with labmda exonuclease buffer.

See also the book Enzymes of molecular biology

and http://www.my-whiteboard.com/excellgen-commonly-used-enzymes/

RecA

Exonuclease III: Double-strand selective 3'->5' exonuclease. Used in a LIC-like procedure in this paper.

Ligases

T4 DNA ligase: Catalyzes the covalent linking of DNA fragments. Requires ATP for enzymatic activity. The activity of T4 DNA polymerase may be measured in Weiss units or Cohesive End Units. From NEB webpage: "A Cohesive End Unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lambda DNA (5´ DNA termini concentration of 0.12 µM (300 µg/ml)) in 20 µl of 1X T4 DNA Ligase Buffer in 30 minutes at 16°C. "

Phospatases

Calf Intestine Phosphatase:

Not readily heat inactivated. Shrimp Alkaline Phosphatase (SAP):

http://www.thermoscientific.com/ecomm/servlet/productsdetail_11152___13318711_-1

Antarctic Shrimp Phosphatase:

• Inactivated by incubation at 65C for 5 min.
• Requires Zn2+ for activity (Reaction buffer supplied).
• Purification not needed before ligation.

See also Bitesize Bio: Should you use calf itestinal alkaline phospatase (CIP) in plasmid cloning?