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Protein Description
Item # 1002
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| Protein : |
E.coli DNA Polymerase I
Large (Klenow ) Fragment, 3'-5' exonuclease (-) |
| Lot # : |
080806BM |
| Concentration : |
40,000 units/ml ; 2.93 mg/ml
|
| Package format : |
50
µl = 2000 units enzyme. Also included are 1.0 ml dilution buffer and 1.0ml 10X reaction buffer.
|
| 10X Reaction
Buffer: |
500mM KPO4 pH 7.0,
60mM MgCl2,
50mM 2-Mercaptoethanol |
| 1X Dilution Buffer: |
50mM KPO4 pH 7.0, 100mM KCl, 1mM DTT, 50% v/v Glycerol |
| Description : |
This protein is an N-terminal truncation of E.coli DNA Polymerase I at amino acid #323 . In this construct, the domain containing the 5 ´- 3 ´ exonuclease activity is deleted, leaving the polymerase function intact. In addition two mutations are introduced (D355A and E357A) which abolish the 3 ´- 5 ´ exonuclease activity (1).
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| Protein Uses : |
Dideoxy sequencing (2), blunt ending restriction fragments, second strand cDNA synthesis for labeling and for use in mutagenesis protocols (3). The enzyme is extensively utilized for labeling of DNA with nucleotide analogs for use as microarray probes (4). |
| Properties : |
The enzyme features strand displacement capability , an error rate of @ 100x10-6 bases and has no intrinsic exonuclease activity.
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Protein Quality Assurance Verification
Item # 1002 |
|
Protein : |
E.coli DNA Polymerase I Large (Klenow ) Fragment,
3'-5' exonuclease (-) , Lot # : 080806BM
|
 |
Concentration : |
40,000 units/ml ; 2.93 mg/ml |
Formulation : |
50mM KPO4 pH 7.0, 100mM KCl, 1mM DTT, 50% v/v Glycerol
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|
Quality Assessment |
Assay |
Result |
| ds endonuclease
|
Degrade
ΦX174 RF |
None
Detected
|
| ss endonuclease |
Degrade M13mp18 |
None
Detected
|
| 5' ss+ds exonuclease |
Removal of labeled nucleotide from 5'
end of a ss or ds oligonucleotide
|
None
Detected
|
| 3' ss+ds exonuclease |
Removal of labeled nucleotide from 3'
end of a ss or ds oligonucleotide |
None
Detected
|
| unit functional assay |
1 unit incorporates 10nMol32P dTTP into
acid insoluble material in 30 minutes at 37° C
using poly dA/dT as substrate |
Pass |
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