Filename:. BLUNTEND.doc Written by: R. Johnson (7/5/97)
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Protocol: Blunt Ending DNA Fragments For Ligation
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Removing 3’ Overhangs Using T4 DNA Polymerase (NEB)
Digest 1-5 µg DNA in a 20 µl reaction. Use a restriction buffer compatible with T4 DNA
Polymerase.
(optional) Check a small aliquot to ensure complete digestion.
Chill reaction on ice, then add 1 µl 2 mM dNTP and 1-2 units T4 DNA Polymerase for
each µg DNA.
Incubate at 12oC for 20 min.
Heat inactivate enzyme at 75oC for 10 min.
Activity in restriction buffers:
NEB 1..........................................60%
NEB 2..........................................100%
NEB 3..........................................100%
NEB 4..........................................100%
Stratagene 0.5X Universal ...........50%
(Note: This protocol will also fill in 5’ overhangs)
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Filling In 5’ Overhangs Using Klenow Fragment
Digest 1-5 µg DNA in a 20 ul reaction.
(Optional step) Check a small aliquot to ensure complete digestion.
Add 0.5 µl 2 mM dNTP and 1 unit Klenow for each µg DNA.
Incubate at 30 oC or RT for 15 min.
Heat inactivate enzyme at 75oC for 20 min, or by phenol-chloroform extraction followed
by alcohol precipitation.
(Note: Klenow Fragment is 50 % active in NEB restriction buffers (1,2,3,4), 100% active in
0.5X Stratagene Universal Buffer (80% in 1X))
(Caution: excessive amounts of enzyme or long reaction times may result in recessed ends due to
the 3’ 5’ exonuclease activity of the enzyme.)
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Removing Single-Stranded Extensions By Mung Bean Nuclease (NEB)
Suspend DNA (0.1 µg/1 µl) in 1X Mung Bean Nuclease Buffer (50mM Sodium acetate
pH 5.0, 30 mM NaCl, 1 mM ZnSO4)
Add 1.0 unit of Mung Bean Nuclease per µg of DNA (Note: enzyme may be diluted in
reaction buffer.)
Incubate at 30 oC for 30 min.
Inactivate the enzyme by phenol-chloroform extraction or by addition of SDS to 0.01%.
Recover the DNA by alcohol precipitation.
Eipper/Mains Protocol Manual
Filename:. BLUNTEND.doc Written by: R. Johnson (7/5/97)
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Blunting Taq DNA Polymerase Generated PCR Fragments
Clean up PCR-generated DNA (Gel purify if desired).
Set up a 100 µl reaction using 25% to 50% of starting DNA material, including 10 µl of
10X Pol I buffer (0.5M Tris pH 7.5, 0.1M MgCl2, 10mM DTT, 0.5 mg/ml BSA, 200 µM
dNTP’s), rATP to 1 mM, and about 10 units each of T4 polynucleotide kinase and DNA
Polymerase I; adjusting final volume to 100 µl with water.
Incubate at 37 oC for 60 min.
Add 1 µl of 0.5M EDTA to stop the reaction.
Clean up DNA using Geneclean (Bio 101) or Qiaex (Qiagen).
Elute DNA in 20-30 µl buffer.
Chemical
DNA pol 1
dNTPs
GeneClean
Klenow polym.
Mung Bean Nuclease
Quiex columns
T4 DNA pol.
T4 polynuc.Kin.
Vendor
New England Bio.
USB
Bio101
Boehringer Mann.
New England Bio.
Quiagen
New England Bio.
New England Bio.
Solutions:
Special notes, common problems, and troubleshooting:
References:
Eipper/Mains Protocol Manual
Catalog #
209S
1001-400
1008404
250S
20021
203S
201S