Yes, spike-in primers have been designed and tested to address the two low coverage areas (amplicons 49 and 57) caused by BA.2 variants (A20055G, G2275G). These primers can be provided free of charge upon request. Please contact info@neb.com. Alternatively, the sequences below can be used for custom primer synthesis from your preferred oligo provider. These spike-in primers should be used with the VarSkip Short v2 primers following the protocols below.
| Primer | Sequence | Target conc. in 1x Pool (µM) |
|---|---|---|
| 57_LEFT_ALT2 | ATG TCT ATG CAG ATT CAT TTG TAA TTA GAG G |
0.4 |
| 49_RIGHT_ALT1 |
CAT TAC GGG CAT TTC TAA ATA AGT CTA C |
0.2 |
Protocols for use of spike-in primers:
For 96-reaction kits:
- Thaw BA.2 Spike-in Mix and VarSkip Short v2 Primer Mix 1.
- Mix and quick spin both.
- Add 1 μl of the BA2 Spike-in Mix to the entire VarSkip Short v2 Primer Mix 1 tube.
- Mix and quick spin spiked VarSkip Short v2 Primer Mix 1.
- Use spiked VarSkip Short v2 Primer Mix 1 for cDNA Amplification protocol.
For 24-reaction kits:
- Thaw BA.2 Spike-in Mix and VarSkip Short v2 Primer Mix 1
- Mix and quick spin both.
- Add 1 μl of BA2 Spike-in Mix to 3 μl 0.1x TE to make a ¼ dilution of the BA.2 Spike-in Mix.
- Add 1 μl of the diluted BA2 Spike-in Mix to the entire VarSkip Short v2 Primer Mix 1 tube.
- Mix and quick spin spiked VarSkip Short v2 Primer Mix 1.
- Use spiked VarSkip Short v2 Primer Mix 1 for cDNA Amplification protocol.