Blunting ends by 3’ overhang removal and 3’ recessed (5’ overhang) end fill-in using T4 DNA Polymerase (M0203)

Steps

Dissolve DNA in any 1X supplemented with 100micromolar (µM) .

Note

T4 DNA Polymerase can be used in T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart® Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. , T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart® Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. , and T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart® Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. as well as T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart® Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. , T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart® Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. , and T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart® Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. and T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart® Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. . Optimal activity is observed in T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart® Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. . BSA supplementation is recommended when using a buffer that does not already contain BSA.

Add 1 unit of T4 DNA Polymerase per microgram DNA.

Incubate 0h 15m 0s at 12°C.

Stop reaction by adding EDTA to a final concentration of 10millimolar (mM).

Note

See references 1, 2 for information on stopping reaction using EDTA and 75°C.

Heat for 0h 20m 0s at 75°C.

Note

CAUTION : Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme.

Abstract

Before start

Steps

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