DNA Polymerase I, Large (Klenow) Fragment

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
M0210S     -20       DNA Polymerase I, Large (Klenow) Fragment M0210SVIAL -20 1 x 0.04 ml 5,000 units/ml   NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
M0210L     -20       DNA Polymerase I, Large (Klenow) Fragment M0210LVIAL -20 1 x 0.2 ml 5,000 units/ml   NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
M0210M     -20       DNA Polymerase I, Large (Klenow) Fragment M0210MVIAL -20 1 x 0.02 ml 50,000 units/ml   NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X

Application Features

• DNA sequencing by the Sanger dideoxy method (2)
• Fill-in of 5´ overhangs to form blunt ends (3)
• Removal of 3´ overhangs to form blunt ends (3)
• Second strand cDNA synthesis
• Second strand synthesis in mutagenesis protocols (4)

Companion Products

• Deoxynucleotide (dNTP) Solution Set
• Deoxynucleotide (dNTP) Solution Mix

Materials Sold Separately

• NEBuffer 2

1. Protocol for blunting ends by 3' overhang removal and 3' recessed end fill-in: 
   • DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 33 μM each dNTP. Add 1 unit DNA Polymerase I, Large (Klenow) Fragment per microgram DNA and incubate 15 minutes at 25°C. Stop reaction by adding EDTA to a final concentration of 10 mM and heating at 75°C for 20 minutes.
2. Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme.
3. When DNA Polymerase I, Large (Klenow) Fragment is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 μl reaction volume is recommended.
4. DNA Polymerase I, Large (Klenow) Fragment is also active in all four NEBuffers and T4 DNA Ligase Reaction Buffer when supplemented with dNTPs.

1. Jacobsen, H., Klenow, H. and Overgaard-Hansen, K. (1974). Eur. J. Biochem.. 45, 623-627.
2. Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.40-5.43. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
4. Gubler, U. (1987). In S.L. Berger and A.R. Kimmel(Ed.), Methods in Enzymology. 152, 330-335. San Diego: Academic Press.
5. Bebenek, K., Joyce, C.M., Fitzgerald, M.P. and Kunkel, T.A. (1990). J. Bio. Chem . 265, 13878-13887.

1. Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210)

• Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

• Blunting Selection Chart
• DNA Polymerase Selection Chart

• NEBcloner^®

1. Which DNA polymerase can I use to blunt DNA?
2. Can I use a DNA polymerase to fill in 3' overhangs or remove 5' overhangs?
3. How can I heat inactivate this DNA polymerase?
4. Can this DNA polymerase be used in labeling and partial fill reactions?
5. What are the main causes of blunting reaction failure?
6. Can this DNA polymerase be used in other NEBuffers for filling in or blunting overhangs?

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.

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]

• M0210M_v1
• M0210S_L_v1

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]

• M0210M_v1_0891509
• M0210S_L_v1_0891509
• M0210S_L_v1_0891512
• M0210S_L_v1_0891603
• M0210M_v1_0891603
• M0210M_v1_0891608
• M0210S_L_v1_0891608
• M0210M_v1_0891703
• M0210S_L_v1_0891703
• M0210M_v1_0891709
• M0210S_L_v1_0891709
• M0210M_v1_0891803
• M0210S_L_v1_0891803
• M0210L_v1_10007540
• M0210L_v1_10025743
• M0210S_v1_10024368
• M0210L_v1_10028508
• M0210M_v1_10028233
• M0210S_v1_10028423
• M0210L_v1_10034214
• M0210M_v1_10034564
• M0210S_v1_10034001
• M0210L_v1_10036837
• M0210S_v1_10037080
• M0210L_v1_10046211
• M0210S_v1_10043965
• M0210S_v1_10050337
• M0210L_v1_10053482
• M0210S_v1_10055962
• M0210L_v1_10057607
• M0210L_v1_10058269
• M0210S_v1_10058172
• M0210L_v1_10062077
• M0210M_v1_10057608
• M0210S_v1_10062081
• M0210L_v1_10065299
• M0210S_v1_10066373
• M0210L_v1_10075466
• M0210S_v1_10075464
• M0210S_v1_10079480
• M0210M_v1_10087408
• M0210L_v1_10088146
• M0210S_v1_10087380
• M0210S_v1_10094981
• M0210L_v1_10100634
• M0210M_v1_10101977
• M0210L_v1_10106852
• M0210S_v1_10107645
• M0210S_v1_10113999
• M0210L_v1_10117205
• M0210M_v1_10117349
• M0210M_v1_10123077
• M0210S_v1_10127909
• M0210L_v1_10136292
• M0210M_v1_10137266
• M0210L_v1_10149552
• M0210S_v1_10150626
• M0210L_v1_10155130
• M0210M_v1_10154966
• M0210L_v1_10162372
• M0210L_v1_10170535
• M0210S_v1_10169279
• M0210S_v1_10172115
• M0210M_v1_10174165
• M0210S_v1_10179255
• M0210L_v1_10179232
• M0210S_v1_10185065
• M0210L_v1_10189289
• M0210S_v1_10197176
• M0210L_v1_10203097
• M0210S_v1_10203992
• M0210S_v1_10207806
• M0210M_v1_10205873
• M0210L_v1_10208202
• M0210S_v1_10213763
• M0210L_v1_10218688
• M0210S_v1_10222867
• M0210L_v1_10226640
• M0210S_v1_10232034
• M0210M_v1_10221782
• M0210S_v1_10238788
• M0210L_v1_10239923
• M0210M_v1_10243114
• M0210L_v1_10249063
• M0210S_v1_10250254
• M0210L_v1_10257142
• M0210S_v1_10257141
• M0210M_v1_10265547
• M0210L_v1_10270932
• M0210S_v1_10270938
• M0210L_v1_10277057
• M0210L_v1_10282654
• M0210S_v1_10281966
• M0210L_v1_10287133
• M0210M_v1_10284955
• M0210L_v1_10297479
• M0210M_v1_10298575
• M0210S_v1_10298576
• M0210S_v1_10306640
• M0210L_v1_10303415
• M0210S_v1_10309951
• M0210L_v1_10313550
• M0210S_v1_10321527
• M0210S_v1_10330851
• M0210L_v1_10329620

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

• DNA Polymerase I, Large (Klenow) Fragment
• NEBuffer™ 2

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