Blunting ends by 3’ overhang removal and 3’ recessed (5’ overhang) end fill-in using T4 DNA Polymerase (M0203)
New England Biolabs
Published: 2022-02-14 DOI: 10.17504/protocols.io.be6ejhbe
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Abstract
Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using T4 DNA Polymerase.
Before start
Steps
1. 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.
Dissolve DNA in any 1X supplemented with 100micromolar (µM) .
Note
2.
Add 1 unit of T4 DNA Polymerase per microgram DNA.
3.
Incubate 0h 15m 0s at 12°C.
4. See references 1, 2 for information on stopping reaction using EDTA and 75°C.
Stop reaction by adding EDTA to a final concentration of 10millimolar (mM).
Note
5. 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.
Heat for 0h 20m 0s at 75°C.
Note
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