Words similar to dttp
Example sentences for: dttp
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To generate the probe of HL60 DNA, a PCR reaction was set up with 0.5 μl of the reassociated DNA, 5 μl of 10x Clontech PCR buffer, 1 μl of dNTPS (10 mM dATP, dTTP, and dGTP each, 6 mM dCTP), 4 μl of FluoroLink Cy3-dCTP (Amersham Pharmacia), 4.5 μl of 10 μM CT primer, 1 μl of Clontech cDNA polymerase and 34 μl of ddH 2 O. In the PCR reaction for the probe of HL60/TLK286 DNA, Cy5-dCTP and TT primer were used instead of Cy3-dCTP and CT primer.
Up to 50 ng of CIP-treated template DNA were incubated for 20 minutes at 37°C in a 10 μL solution containing 20 U TdT (NEB), 0.2 M potassium cacodylate, 25 mM Tris-HCl pH 6.6, 0.25 mg/ml BSA, 0.75 mM CoCl 2 , 4.6 μM dTTP and 0.4 μM ddCTP.
Interestingly, the p66 INS/p51 INSmutant carrying insertion in both the subunits did not display the same reduction in dTTP binding affinity.
To generate labeled single-stranded cDNA target, 10 μg total RNA from rat heart or brain (Clontech, Palo Alto, CA) was reverse transcribed for 2-3 h at 42°C in the presence of 6 μg random primers (Invitrogen/Life Technologies), 1x first-strand synthesis buffer (Invitrogen/Life Technologies), 10 mM DTT, dNTP mix (25 mM dATP, 25 mM dCTP, 25 mM dGTP, 15 mM dTTP, 10 mM amino allyl-dUTP), and 200 units Superscript II reverse transcriptase (Invitrogen/Life Technologies).
The following reagents were added: 6 μl of 5X first strand buffer, 3 μl 0.1 M DTT, 0.7 μl 50X dNTP (25 mM dATP, dCTP, dGTP and 10 mM dTTP), 3 μl 1 mM Cy3-dUTP or Cy5-dUTP and 1.7 μl Superscript™ II (200 U/μl).
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