1. Is 4BB™ SunScript^® RT compatible with other DNA polymerase to use it in a single-tube reaction?

There is no problem to use our 4BB™ SunScript^® enzyme together with other DNA Polymerase, it should work. In fact, we have a One-Step RT-PCR kit in which we combine 4BB™ SunScript^® with a High Fidelity Taq Polymerase to perform both reverse transcription and amplification in the same single tube. With 4BB™ SunScript^® you can fix the reverse transcription temperature from 50ºC up to 85ºC according to your requirements so you have a wide range of possible temperatures.

2. Would 4BB™ SunScript^® work in a buffer containing TrisHCl 20mM (pH8.0) and 80mM KCl?

Yes, 4BB™ SunScript^® should perfectly work in it.

3. Would 95ºC for 5min be OK for deactivation the enzyme?

We recommend 10 min 95ºC for complete inactivation but it can be reduced to 5 min without any problem.

4. How do you get a Poly T primer annealed at 85ºC?

Annealing of PolyT at 85ºC: in SunScript protocol we recommend to perform a previous annealing step at 68ºC for 5 min. Then, the reaction temperature (from 50ºC to 85ºC) can be chosen for the cDNA synthesis step. It is supposed that the RT is already bound to the PolyT annealed to the RNA in the very first moment in which the reaction temperature reaches 85ºC, and the high efficiency and speed of 4BB™ SunScript^® allows to perform the reverse transcription reaction despite the annealing is broken soon after.

 5. What is the minimum template material required for the 4BB™ Sunscript^® kits?

For regular kits of 4BB™ SunScript^® the minimum recommended amount of template is 10 pg Total RNA or PolyA RNA. For the One-Step RT-PCR kit the systems works from starting total RNA amounts of 1 ug to pg. For the qPCR kit is 10 pg.

6. Would you be able to tell me how many 4BB™ SunScript^® units per reaction is included in your 4BB™ SunScript^® One Step RT-PCR Kit?

A one step RT-PCR reaction can perfectly work with only 7U 4BB™  SunScript^® minus.