Hi to all,
We successfully created in our lab a new RT-PCR system, designed for amplification of the S1 segment of Infectious bronchitis virus (IBV). Yet, we obtained the end point PCR product only by using RNA from enriched allantois fluid, derived by inoculating SPF embryonated eggs. What is the best one-step RT-PCR kit/enzyme that we can use to enhance the segment using RNA directly extracted from tracheal/cloacal swabs? according to what I read, the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is the best option, due to the increased sensitivity and the broad temperature range of the cDNA synthesis (We currently use the Verso one-step RT-PCR kit).
Will appreciate any insight,
Ido
I often work with AgPath-ID™ One-Step RT-PCR master mix, and
Luna® Universal One-Step RT-qPCR without any problem. But you can use SuperScript™ III One-Step RT-PCR System if you have it.
Ido From
The best way of end-point amplification of target region using RNA directly extracted from tracheal/cloacal swabs is making complementary DNA (Step 1) and amplify target region from complementary DNA (Step 2).
One-step RT-PCR system can lead to lower yields or efficiency, more complicated to troubleshoot, and does not provide a stock of cDNA for future use. Since RNAs are unstable so, you can reverse transcribe them immediately and perform PCR later (Two-step PCR System).
Still if you want to use One-Step RT-PCR system, you may use following kits;
1. QIAGEN OneStep RT-PCR Kit, https://www.qiagen.com/us/products/discovery-and-translational-research/pcr-qpcr-dpcr/pcr-enzymes-and-kits/one-step-rt-pcr/qiagen-onestep-rt-pcr-kit/
2. SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (you mentioned).
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