It’s possible to use RNA template with a TwistAmp® kits just by adding a suitable reverse transcriptase (RT) when setting up a reaction. The only difference between a TwistAmp®basic/nfo, and a TwistAmp® basic/nfo RT kits are the addition of an M-MLV RT to the freeze dried reaction. If an RT that works at 37–42 °C is added to RPA chemistry then RNA can be reverse transcribed and the cDNA produced and amplified all in one step. The complementary DNA sequence will be that of the RNA added. We recommend using similar RT amounts to that of a PCR reaction of the same volume, according to your chosen manufacturer’s instructions. We also suggest that you run reactions at 40°C and delay agitation of the reaction by a minute to give the RT time to work, otherwise you just run the reaction as you would a normal TwistAmp® reaction (it’s a one-step process). It’s also advisable to add RNase Inhibitor to any reaction where RNA is the target material. Any commercially available RNase Inhibitor will be suitable.
The one we used prior to making our own RT can be found here:https://www.thermofisher.com/order/catalog/product/EP0441
If we were to try and use it again, we would do a titration to determine optimal quantity to add in the new formulation reactions (we haven’t used it since we changed the formulation of the reactions a couple of years ago).
TwistAmp Basic Kit
(TABAS03KIT)
| 50uL, 96reactions | Recombinase, Polymerase, SSB etc | KRW924,000(+VAT) |
M-MLV H-2.0
(APWL06M1)
| 20,000 U | M-MLV (H-) 2.0 is a Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase with RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity. M-MLV (H-) 2.0 has no 3´→5´ exonuclease activity nor RNase H activity. | KRW330,000(+VAT) |
Murine RNase Inhibitor
| 2000U/10000U/20000U | Murine RNase Inhibitor is a recombinant murine-derived RNase inhibitor expressed and purified in E. coli. It exhibits high affinity and binds non-covalently to RNase A, B, or C in a 1:1 ratio, thereby inhibiting the activity of these enzymes and protecting RNA from degradation. |
|
It’s possible to use RNA template with a TwistAmp® kits just by adding a suitable reverse transcriptase (RT) when setting up a reaction. The only difference between a TwistAmp®basic/nfo, and a TwistAmp® basic/nfo RT kits are the addition of an M-MLV RT to the freeze dried reaction. If an RT that works at 37–42 °C is added to RPA chemistry then RNA can be reverse transcribed and the cDNA produced and amplified all in one step. The complementary DNA sequence will be that of the RNA added. We recommend using similar RT amounts to that of a PCR reaction of the same volume, according to your chosen manufacturer’s instructions. We also suggest that you run reactions at 40°C and delay agitation of the reaction by a minute to give the RT time to work, otherwise you just run the reaction as you would a normal TwistAmp® reaction (it’s a one-step process). It’s also advisable to add RNase Inhibitor to any reaction where RNA is the target material. Any commercially available RNase Inhibitor will be suitable.
The one we used prior to making our own RT can be found here:https://www.thermofisher.com/order/catalog/product/EP0441
If we were to try and use it again, we would do a titration to determine optimal quantity to add in the new formulation reactions (we haven’t used it since we changed the formulation of the reactions a couple of years ago).
(TABAS03KIT)
(APWL06M1)
M-MLV (H-) 2.0 is a Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase with RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.
M-MLV (H-) 2.0 has no 3´→5´ exonuclease activity nor RNase H activity.
Murine RNase Inhibitor is a recombinant murine-derived RNase inhibitor expressed and purified
in E. coli. It exhibits high affinity and binds non-covalently to RNase A, B, or C in a 1:1 ratio,
thereby inhibiting the activity of these enzymes and protecting RNA from degradation.