商品编号


P7070L



品牌


Enzymatics



公司


Enzymatics,inc



公司分类


Ultrapure Polymerases



Size

6,000 U

商品信息

Terminal deoxynucleotidyl Transferase (TdT)


Product Description

Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyzes the addition of deoxynucleotides to the 3′ hydroxyl terminus of single or double stranded DNA molecules. The presence of 1 mM Co
2+
stimulates the tailing of the 3′-ends of DNA fragments. This construct is sold as an N-terminal truncation of the terminal transferase gene attached to an N-terminal fusion tag.

Source of Protein
An?
E. coli
?strain that carries the cloned terminal transferase gene from calf thymus.

Supplied in?
50 mM KPO
4
100 mM NaCl
1.0 mM DTT
0.1 mM EDTA
0.1% Triton X-100
50% Glycerol
pH 7.3 @ 25°C

Supplied With
B0120 (10X Green Buffer)
B0220 (2.5 mM CoCl
2
)

10X Green Buffer (B0120)
200 mM Tris-Acetate
500 mM Potassium Acetate
100 mM Magnesium Acetate
pH 7.9 @ 25°C

Unit Definition
1 unit is defined as the amount of polymerase required to convert 1 nmol of dTTPs into acid insoluble material in 1 hour at 37°C.



Quality Control Analysis

Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 50 ?L reactions containing Oligo dT 20mer DNA, 1X reaction buffer, 0.25mM CoCl
2
?
3
H-dTTP and 100 ?M dTTPs. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using the method of Sambrook and Russell?
(Molecular Cloning, v3, 2001, pp. A8.25-A8.26)
.

Protein Concentration (OD
280
) Measurement
A 2.0 ?L sample of TdT was analyzed at OD
280
?using a
Nanodrop
ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (
Pierce
Cat #23209 and blanked with TdT storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 88,190 and molecular weight of 82,598 Daltons.

SDS-Page (Physical Purity Assessment)
2.0 ?L of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW
Marker
and 2.0 ?L of a 1:100 dilution of the sample. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.



Contamination Tests

Single-Stranded Exonuclease Activity
A 50 ?l reaction containing 10,000 cpm of a r
ADI
olabeled single-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in less than 5.0% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity
A 50 ?l reaction containing 5,000 cpm of a r
ADI
olabeled double-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.

Double-Stranded Endonuclease Activity
A 50 ?l reaction containing 0.5 ?g of pENZuC DNA and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in no visually discern
IBL
e conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli
?16S rDNA Contamination Test
Replicate 5uL samples of enzyme solution were denatured and screened in a TaqMan qPCR assay for the presence of contaminating?
E.coli
?genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (C
t
) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control C
t
?values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.


View PDF Poster Instructions FAQ


Product Information



Terminal deoxynucleotidyl Transferase (TdT)


Part Number
P7070L
Price
$363
Concentration
20,000 U/ml
Unit Size
6,000 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>99%
Specific Activity
27,400 U/mg
SS Exonuclease
200 U <5.0% released
DS Exonuclease
200 U <1.0% released
DS Endonuclease
200 U = No conversion
E.coli
DNA Contamination
200 U <10 copies





* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.

Usage Instructions

Non-templated addition of dNTPs to 3′ termini of DNA:
Reaction Set-Up:




Amount

Description

Final Concentration



5 ?L

10X Green Buffer (B0120)

1X



X ?L

10 pmol DNA termini (10-100 ng)

1-10 ng/?L



x ?L

Deoxynucleotide solution

200 ?M



1 ?L

Terminal Transferase (20 U/ ?L)

30 U/?L



X ?L

Type I Water

N/A



50 ?L

Total Volume




?


Incubate at 37°C for 30 minutes.

Inactivate the TdT and stop the reaction by heating to 70°C for 10 minutes.


Notes

Co
2+
increases the nucleotide incorporation efficiency of pyrimidines, and at blunt and 3′ recessed ends. However, the addition of dNTPs to 3′-overhanging ends is more efficient than with 3′-recessed or blunt ends. TdT requires a free 3′-hydroxyl group in order to make a non-templated nucleotide addition.

With limited efficiency, Terminal Transferase will incorporate ribonucleotides, biotinylated, and dideoxynucleotides in the presence of Co
2+
.

Terminal Transferase incorporates dATP and dTTP with a 5-fold higher efficiency than dCTP and dGTP, as evidenced by the following K
m
values for nucleotides:




Base

K
m



dATP

100 ?M



dTTP

100 ?M



dCTP

500 ?M



dGTP

500 ?M




References


Deng, G.R. and Wu, R. 1983 Meth. Enzymol. 100:96-116.




Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for admi
NIST
ration to humans or animals. SDS sheets relevant to this product are available upon request.