The CleanAmp™ dNTPs have a half life of 5 minutes at 95°C in PCR buffer. At 92°C the deprotection rate will be slightly slower however this temperature should allow deprotection of enough of the CleanAmp™ dNTPs to work in your PCR. If needed, you could extend the time of the initial denaturation. Please contact us if you have any additional questions.
Thank you for your interest in our poster, Pushing the Limits of PCR, qPCR and RT-PCR Using CleanAmp™ dNTPs. An activation step is not required for RT-PCR as the CleanAmp™ dNTPs are activated by the combination of heat at 47°C and the acidification of Tris Buffer with increasing temperature. This releases enough dNTPs to generate a cDNA during the RT reaction. The slower activation helps decrease off-target reaction during both the RT and PCR steps.
I recommend using the conditions shown in Figure 9 of the poster which I have included below for your convenience. Of note, we recommend using a traditional 3-step thermocyling protocol, as it will provide more consistent performance than a faster 2-step protocol.
RT-PCR Conditions: Buffer (50 mM Tris-HCl; 75 mM KCl; 3 mM MgCl2), 0.4 mM CleanAmp™ dNTPs, 0.5 µM Forward Primer, 1.0 µM Reverse Primer, 100 U M-MLV (RNase H-) RT, 2.5 U Taq polymerase, 0.032-500 ng Target RNA, 10 mM DTT, 0.4 U/µL RNase Inhibitor, 25 µL reaction.
RT-PCR Cycling Conditions: 47°C (30 min); 94°C (10 min); [94°C (15 sec), 62°C (30 sec), 72°C (1 min)] 40X; 72°C (5 min)
Sabrina Shore, MS
Thank you for contacting TriLink. I am not aware of any specific enzyme which is known to incorporate 5-carboxy-dCTP however; since different analogs incorporate with different efficiencies, I recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions. Alternatively, decreasing the elongation temperature, increasing elongation time in PCR cycle, increasing concentration of dGTP and 5-carboxy-dCTP or adjusting the pH may support incorporation. Please keep in mind this modification is sensitive to pH and should be kept in basic conditions. Do not hesitate to contact us if you have any further questions.