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DNA Pol I, Large (Klenow) fragment was originally derived as a proteolytic product of E. coli DNA polymerase. It retains polymerase activity, but lacks both 5’ —> 3’ and 3' —> 5’ exonuclease activity.
- Generates probes using random primers
- Dideoxy sequencing
- Random priming labeling
- Moderate strand displacement activity
- Not suitable for generating blunt ends
- Product Information
Highlights
- Isolated from a recombinant source
- Generates probes using random primers
- Dideoxy sequencing
- Supplied with 10X Reaction Buffer
- Moderate strand displacement activity
Product Source
AnE. colistrain containing a plasmid with a fragment of theE.colipolA (D355A, E357A) gene starting at codon 324.Reagents Supplied
The following reagents are supplied with this product:
| NEB # | Component Name | Component # | Stored at (°C) | Amount | Concentration |
| M0212S | -20 |
| Klenow Fragment (3'→5' exo-) | M0212SVIAL | -20 | 1 x 0.04 ml | 5,000 units/ml | |
| NEBuffer™ 2 | B7002SVIAL | -20 | 1 x 1.25 ml | 10 X |
| M0212L | -20 |
| Klenow Fragment (3'→5' exo-) | M0212LVIAL | -20 | 1 x 0.2 ml | 5,000 units/ml | |
| NEBuffer™ 2 | B7002SVIAL | -20 | 1 x 1.25 ml | 10 X |
| M0212M | -20 |
| Klenow Fragment (3'→5' exo-) | M0212MVIAL | -20 | 1 x 0.02 ml | 50,000 units/ml | |
| NEBuffer™ 2 | B7002SVIAL | -20 | 1 x 1.25 ml | 10 X |
Application Features
- Random priming labeling
- DNA sequencing by the Sanger dideoxy method (2)
- Second strand cDNA synthesis
- Second strand synthesis in mutagenesis protocols (3).
Properties & Usage
Unit Definition
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.
Reaction Conditions
1X NEBuffer™ 2
Incubate at 37°C
1X NEBuffer™ 2
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
1 mM DTT
(pH 7.9 @ 25°C)
Storage Buffer
25 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C
Heat Inactivation
75°C for 20 minutes
Molecular Weight
Theoretical: 68000 daltons
5' - 3' Exonuclease
No
3' - 5' Exonuclease
No
Strand Displacement
+++
Unit Assay Conditions
1X NEBuffer 2, 33 µM dNTPs including [3H]-dTTP and 70 µg/ml denatured herring sperm DNA.
Error Rate
~ 100x10-6bases
Companion Products
- Deoxynucleotide (dNTP) Solution Set
- Deoxynucleotide (dNTP) Solution Mix
- Monarch® Plasmid Miniprep Kit
- Monarch® DNA Gel Extraction Kit
Materials Sold Separately
- NEBuffer 2
Notes
- Klenow Fragment (3´→ 5´ exo-) is not suitable for generating blunt ends because it lacks the 3´→ 5´ exonuclease necessary to remove non-templated 3´ additions.
- Klenow Fragment (3´→ 5´ exo-) is also active in all four NEBuffers when supplemented with dNTPs.
- When Klenow Fragment (3´→ 5´ exo-) is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 µl reaction volume is recommended.
References
- Derbyshire, V. et al. (1988). Science. 240, 199-201.
- Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
- Gubler, U. (1987). In S.L. Berger & A.R. Rimmel(Ed.), Methods in Enzymology. 152, 330-335. San Diego: Academic Press.
- Bebenek, K., Joyce, C.M., Fitzgerald, M.P. and Kunkel, T.A. (1990). J. Biol. Chem.. 265, 13878-13887.
Protocols
- A-Tailing with Klenow Fragment (3'→5' exo-)
Usage Guidelines
Web Tools
FAQs
- Which DNA polymerase can I use to blunt DNA?
- Can I use a DNA polymerase to fill in 3' overhangs or remove 5' overhangs?
- How can I heat inactivate this DNA polymerase?
- Can this DNA polymerase be used in labeling and partial fill reactions?
- Can this DNA polymerase be used in other NEBuffers for filling in or blunting overhangs?
Citations
Quality Control Assay
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
- M0212M_v1_0351509
- M0212S_L_v1_0351509
- M0212M_v1_0351603
- M0212S_L_v1_0351603
- M0212S_L_v1_0351608
- M0212S_L_v1_0351503
- M0212M_v1_0351608
- M0212M_v1_0351703
- M0212S_L_v1_0351703
- M0212M_v1_0351709
- M0212S_L_v1_0351709
- M0212M_v1_0351803
- M0212S_L_v1_0351803
- M0212M_v1_10017021
- M0212L_v1_10018714
- M0212M_v1_10018721
- M0212S_v1_10018719
- M0212L_v1_10029069
- M0212M_v1_10029432
- M0212L_v1_10034234
- M0212S_v1_10034369
- M0212L_v1_10034612
- M0212S_v1_10041846
- M0212S_v1_10044656
- M0212S_v1_10045792
- M0212M_v1_10041843
- M0212L_v1_10045791
- M0212S_v1_10048309
- M0212M_v1_10050374
- M0212S_v1_10050369
- M0212L_v1_10051082
- M0212L_v1_10055659
- M0212M_v1_10054193
- M0212S_v1_10056308
- M0212L_v1_10058065
- M0212M_v1_10058006
- M0212S_v1_10061408
- M0212L_v1_10063550
- M0212S_v1_10063557
- M0212M_v2_10065982
- M0212L_v2_10069550
- M0212L_v2_10073009
- M0212M_v2_10073012
- M0212S_v2_10073010
- M0212L_v2_10075647
- M0212L_v2_10083165
- M0212M_v2_10085113
- M0212S_v2_10084994
- M0212L_v2_10088157
- M0212L_v2_10094182
- M0212S_v2_10091536
- M0212L_v2_10098035
- M0212M_v2_10097440
- M0212S_v2_10098602
- M0212M_v2_10102441
- M0212L_v2_10106903
- M0212S_v2_10106902
- M0212L_v2_10111035
- M0212S_v2_10114921
- M0212M_v2_10122259
- M0212L_v2_10131912
- M0212L_v2_10119756
- M0212S_v2_10135768
- M0212L_v2_10138879
- M0212L_v2_10145961
- M0212M_v2_10146272
- M0212L_v2_10152517
- M0212S_v2_10159715
- M0212M_v2_10160172
- M0212L_v2_10162623
- M0212M_v2_10163997
- M0212L_v2_10167677
- M0212M_v2_10169282
- M0212L_v2_10172109
- M0212S_v2_10174172
- M0212L_v2_10177214
- M0212L_v2_10184720
- M0212S_v2_10185361
- M0212L_v2_10191591
- M0212M_v2_10201726
- M0212S_v2_10202690
- M0212L_v2_10203804
- M0212S_v2_10206296
- M0212L_v2_10208716
- M0212M_v2_10206925
- M0212L_v2_10218704
- M0212L_v2_10221933
- M0212S_v2_10215595
- M0212S_v2_10223545
- M0212L_v2_10230349
- M0212L_v2_10236035
- M0212M_v2_10234145
- M0212S_v2_10236860
- M0212L_v2_10239526
Safety Data Sheets
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