Fermentas offers a complete range of dNTP, modified dNTP, ddNTP and NTP solutions for all molecular biology applications.
Deoxyribonucleoside Triphosphates
Fermentas PureExtreme® dNTPs are supplied as aqueous solutions at pH 7.0, available individually or as a set of the four dNTPs at 100 mM concentration. Fermentas also offers dNTP mixes at convenient concentrations for direct use in PCR and other common molecular biology applications.
The neutral pH of dNTP solutions ensures high stability of a product during long-term storage:
Fermentas dNTPs are stable for years at -20°C.
- 90-95% of dNTPs are stable after 7 weeks at room temperature.
- 85-90% of dNTPs remain in triphosphate form after 30 cycles of PCR (1 min at 94°C; 3 min at 72°C). This ensures a high amplification yield.
To meet the PureExtreme® standard, all Fermentas nucleotides pass HPLC, RNase assay and Labeled Oligonucleotide (LO) test (see fig.1 below). In addition, all dNTPs are functionally tested in PCR with Taq and Pfu DNA Polymerases.
Figure 1. Purity evaluation of Fermentas dNTPs: LO test.
The LO test allows identification of trace endo-, exodeoxyribonuclease and phosphatase contaminants in dNTP preparations at levels often not detected by conventional methods. The test is performed using two synthetic radiolabeled oligonucleotides (17-mers single and double stranded) as substrates. Each substrate is incubated for 4 hours with each commercial dNTP solution in RT buffer (50 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2 and 10 mM DTT). The reaction products are analyzed in denaturing 20% PAGE followed by phosphor imaging. The dNTP conforms to the quality criterion if there are no visible traces of oligonucleotide degradation.
M - partial enzymatic digestion of 17-mer oligonucleotide;
K1 - control 17-mer oligonucleotide in water, nuclease-free;
K2 - control 17-mer oligonucleotide in buffer;
A - test of dATP;
G - test of dGTP;
T - test of dTTP;
C - test of dCTP.Features
Functionally tested in PCR with Taq and Pfu DNA Polymerases.
- Purity >98% by HPLC.
- High stability.
- Free of other nucleotide contamination.
- Free of endo-, exodeoxyribonuclease, ribonuclease and phosphatase contamination (see fig.1 above and fig.2 below).
Figure 2. Purity evaluation of Fermentas dNTPs: incubation with RNA transcripts.
Three different Fermentas commercial dNTP solutions (in a final dNTP concentration of 1 mM) were incubated with 1 µg of RNA transcripts 200 b and 1 kb long for 4 hours at 37°C in 20 µl reaction volume. There are no visible traces of RNA transcript degradation after the reaction.
M - RiboRuler™ Low Range RNA Ladder, ready-to-use;
K1 - control RNA transcript 1 kb long;
K2 - control RNA transcript 200 b long;
A - test of 2 mM dNTP Mix;
B - test of 10 mM dNTP Mix;
C - test of dNTP Set.Applications
Fermentas dNTPs can be used with confidence in the most demanding applications, including PCR, long PCR, cDNA synthesis, RT-PCR, DNA labeling and DNA sequencing where high quality of dNTPs is as important as high quality of an enzyme.
Performance of PureExtreme® dNTPs in RT-PCR and long PCR is illustrated in fig.3 and fig.4.
a - primed with oligo(dT)18
b - primed with random hexamer
Figure 3. Performance of Fermentas dNTPs in highly sensitive two step RT-PCR.
Varying amounts of total mouse liver RNA were used in a first strand cDNA synthesis reaction with RevertAid™ H Minus M-MuLV Reverse Transcriptase. Then 2 µl of the reverse transcription reaction mixture were used in subsequent PCR with Taq DNA Polymerase and a primer pair specific to the GAPDH (glyceraldehyde phosphate dehydrogenase) gene.
M - GeneRuler™ 100 bp Plus DNA Ladder.
Figure 4. Amplification of long DNA fragments using Fermentas dNTPs.
Long DNA fragments amplified in PCR mix containing 2.5 u of Long PCR Enzyme Mix, 1.25ng of lambda DNA, 0.2 mM of each dNTP and 1.5 mM of MgCl2 in 50 µl reaction volume.
M - Lambda Mix Marker.
| Home | Search | Contacts | Order | Catalog | Support |
Updated vasario 01, 2008 10:42
