What are the differences between Version 1 (V1) and Version 2 (V2)?

CORALL V2 Kits come with two reverse transcription chemistries: RTM and RTL. RTL Chemistry can be used to increase the average library size to approximately 550bp (blue traces) compared to RTM Chemistry which generates libraries with an average size of approximately 350bp (red traces).

Please note that two protocols are provided (i.e., Short Size Inserts (RTM) protocol and Long Size Inserts (RTL) protocol), depending on the reverse transcription chemistry to be used.

An update has been made to our PCR system, where PCR and E3 have been replaced with PM and PE. With this, a new PCR program has also been implemented (see below).

Below are a list of protocol changes, represented in green font:

Reverse Transcription

Prepare a mastermix of 14 µl RTM or RTL and 1 µl DSP per sample. Mix well.
Add 15 µl of RTM or RTL / DSP mastermix to 10 µl RNA sample. Mix well.
Incubate for 3 min at 94 °C, then 15 min at 16 °C for RTM.
Incubate for 1 min at 94 °C, then 15 min at 16 °C for RTL.
Prepare a mastermix of 4 µl RTM or RTL and 1 µl E1 per sample. Mix well.
Add 5 µl RTM or RTL / E1 and mix well.
Incubate: 10 min at 25 °C, 40 min at 37 °C, 10 min at 42 °C, cool to 25 °C. Proceed immediately to purification!

Endpoint PCR

Prepare a mastermix with 7 μl PM and 1 μl PE per reaction. Mix well.
Add 8 μl of PM / PE mastermix to 17 μl of each purified library.
Add 10 µl of one Unique Dual Index Primer pair (UDI12A_0001-0384, or UDI12B_0001-0096) to each sample.
Conduct 11 – 25 cycles of PCR (as determined by qPCR assay) with the following program:
- Initial denaturation at 95 °C for 60 seconds;
- 11 – 25 cycles of
  - 95 °C for 15 seconds,
  - 60 °C for 15 seconds, and
  - 72 °C for 60 seconds,
- and a final extension of 72 °C for 6 minutes. Hold at 10 °C.