Yes, spike-in primers have been designed and tested to address low coverage areas caused by KP and JN variants. 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.
|
VSSv2f Spike-in Mix |
Primer |
Sequence |
Target Concentration in 1xPool (µM) |
|---|---|---|---|
|
1 |
varskip-0317-1_1_LEFT_ALT3 |
ATCTCTTGTAGATCTGTTCTCTAAACG |
0.5 |
|
1 |
varskip-0317-1_05_RIGHT_ALT2 |
GACAATTTCACAAGCACAGGTTGAG |
0.5 |
|
1 |
varskip-0317-1_07_LEFT_ALT2 |
CCTCTAAAAGCCCCAAAAGAAATTATC |
0.5 |
|
1 |
varskip-0317-1_09_RIGHT_ALT2 |
GTTTACTTTCAGTTATAAATGGCTTAACTTC |
0.5 |
|
1 |
varskip-0317-1_11_LEFT_ALT2 |
GTGGCACTACTGAAATGCTAGC |
0.5 |
|
1 |
varskip-0317-1_13_RIGHT_ALT2 |
TTCATAAGAAAGTGTGCCCATGTAC |
0.5 |
|
1 |
varskip-0317-1_15_RIGHT_ALT1 |
GCTCCAAAGACAACGTATACACC |
0.5 |
|
1 |
varskip-0317-1_35_LEFT_ALT2 |
GATGATTATTTCAATAAAAAGGACTGGTATG |
0.5 |
|
1 |
varskip-0317-1_45_RIGHT_ALT2 |
ATTGATCTCCAGGCGGTGGTTT |
0.5 |
|
1 |
49_R_ALT2 |
GTAATAAGAACACCATTACGGGCAT |
0.5 |
|
1 |
varskip-0317-1_53_RIGHT_ALT2 |
TGAGGATCTGAAAACTTTGTCAGG |
0.5 |
|
1 |
55_L_ALT1 |
CCTACTTATTGTTAATAACGCTACTAATGTT |
0.5 |
|
1 |
varskip-0317-1_57_LEFT_ALT6 |
CTCTGCTTTACTAATGTCTATGCAGA |
0.5 |
|
2 |
varskip-0317-1_24_LEFT_ALT2 |
GCCTTTAATACTTTACTATTCCTTATGTC |
0.5 |
|
2 |
varskip-0317-1_28_RIGHT_ALT2 |
AGGCCAAAGTAACAAGTACAAAAATAGC |
0.5 |
|
2 |
varskip-0317-1_32_RIGHT_ALT2 |
CTGTTATTGCCTGACCAGTACC |
0.5 |
|
2 |
varskip-0317-1_34_RIGHT_ALT2 |
ATCACAGAATTGTACTGTTTTTAACAAAGC |
0.5 |
|
2 |
varskip-0317-1_54_RIGHT_ALT2 |
CTTCAAGGTCCATAAGAAAAGGCT |
0.5 |
|
2 |
56L_ALT1 |
TTATTATGTGGGTTATCTTCAACCTAGG |
0.5 |
|
2 |
varskip-0317-1_56_RIGHT_ALT2 |
CAGCCTGTAAAATCATCTGGTAATTTAT |
0.5 |
Protocols for use of spike-in primers:
For 96-reaction kits:
- Thaw VSSv2f Spike-in Mix 1, VSSv2f Spike-in Mix 2, VSSv2 Primer Mix 1. and VSSv2 Primer Mix 2.
- Mix and quick spin all four tubes.
- Add 3 μl of VSSv2f Spike-in Mix 1 to VSSv2 Primer Mix 1, vortex, spin-down, place on ice.
- Add 2.5 μl of VSSv2f Spike-in Mix 2 to VSSv2 Primer Mix 2, vortex, spin-down, place on ice.
- Use spiked VarSkip Short v2 Primer Mix 1 and 2 for RT-PCR protocol.
For 24-reaction kits:
- Thaw VSSv2f Spike-in Mix 1, VSSv2f Spike-in Mix 2, VSSv2 Primer Mix 1. and VSSv2 Primer Mix 2.
- Mix and quick spin all four tubes.
- Add 2 μl of VSSv2f Spike-in Mix 1 to 2 μl 0.1x TE to make a ½ dilution of the VSSv2f Spike-in Mix 1.
- Add 1.5 μl of diluted VSSv2f Spike-in Mix 1 to VSSv2 Primer Mix 1, vortex, spin-down, place on ice.
- Add 2 μl of VSSv2f Spike-in Mix 2 to 2 μl 0.1x TE to make a ½ dilution of the VSSv2f Spike-in Mix 2.
- Add 1.25 μl of diluted VSSv2f Spike-in Mix 2 to VSSv2 Primer Mix 2, vortex, spin-down, place on ice.
- Use spiked VarSkip Short v2 Primer Mix 1 and 2 for RT-PCR protocol.
