Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:139429332+139429494 163bp AGGCAAGTATCCTGCTCCTG AAAAGGACAATCACAGTGCATC
Mut= 168 bp
Wt= 163 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case and insertion with carrots ^g^ (TTTTTTAG insertion):
TTAGAAATACCTGAATGAGTACTTTTTTTTTTTTTTTTAAACAACCTAAGCATTTGAGGGAAAGTATACTTTAAGCATTAAGCTCAAAGTGCTGGGTCTTTGAGGTTCATTTAGTTAGAAATTTAGGTAAGTAGTTTTCTCTTTAGTT^ccctatgtgaatttgggtatatatagtaaggttattaaatggtgccttttcattgcaatgcgaagtttccgtcaaagcagagcagatacgttgaactcaattttcttctctattgtagggaaatatgagagccacacccgtgtgcacacaggtgagaagccttttgagtgtgatatttgtcaccagcgttattcgacaaagtctaacttaactgttcacagaaagaagcacagtaacgaagtagagtttcataggaaggagcacaagtgcccttactgtaacaagctgcacgcaagcaagaagacactggccaagcacgtcaagaggcaagtatcctgctcctggcgtcacacaagtaacaaatgaccaaacaaaagcaaatggcagctcgttcatgttttttctcactgttgatgaggccctttggcagt^GCAACAAGTATATGGCTGATAATGTGAAGTTTAAGATGCACTGTGATTGTCCTTTTTTTTTTTTAAGATTTTTATTTATTATTATTCATTAGTACACTGTAGCTGTCTTCAGATGCACCAGAAGAGGGCGACAGATATCATTACGGGT
This mutation is a 431 bp deletion beginning at Chromosome 1 position 139,429,008 bp and ending after 139,429,438 bp (GRCm38/mm10) . In addition, there is an 8 bp insertion (CTAAAAAA) at the deletion site..
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44027 | AAA AGG ACA ATC ACA GTG CAT C | Common | A | |||
44029 | Fluorophore-1 | CTC ACT GTT GAT GAG GCC CT | Quencher-1 | WT Probe | ||
45397 | Fluorophore-2 | TGG GTC TTT GAG GTT CAT TTA GTT AG | Quencher-2 | MUT Probe | ||
45398 | CCT AAG CAT TTG AGG GAA AGT | Mutant Forward | A | |||
45399 | AGG CAA GTA TCC TGC TCC TG | Wild type Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44027 | 0.40 uM |
45398 | 0.40 uM |
45399 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |