Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 110 bp
Wild Type = 120 bp
>chr5:21648737+21648856 120bp TGGGAAATAATGCAGCCTTC GCTGTGAGCACAAGTGGCTA
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case; insertions with carrots ^g^ (g insertion):
AAAGTGTAAGCAGGCGTTCCCACACACTGCACCTCCCTGGTCCTCAGTAGC^ccccccc^gcctcccctcccacgggccagcctccatggctgacaccacttgcatttttaaagccatgaccttccttcttcctcttctcccatctccctcttcctagcagatgccaagatttgtagcttgcctgtcctgtggtttttctcttaacacatgaaaattgtccatgacctttaaaaagccaaacaactgtgcccagtatcagagtatatgtttgaacatttcacacattaacaataaaacacattactggtgagtttcaatgaattctaaatggcttccttcttatgcagaagacctaaccgatggctcctatgacgatatcttaaatgcagagcagcttaagaaacttctgtatctgctggagtcaaccgacgatcctgtcattactgaaaaggccttggtcaccttgggaaataatgcagccttctccactaaccaggtaagcctcctctttccacacaagtagctgtggccccattctgaaCTCTTAGCTATCCTCATATGGCATAGCCACTTGTGCTCACAGCAGTGTTTG
This mutation is a 482 bp deletion beginning at Chromosome 5 position 21,648,332 bp and ending after 21,648,813 bp (GRCm38/mm10).
There is a 7 bp (CCCCCCC) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66324 | TGG GAA ATA ATG CAG CCT TC | Wild type Forward | A | |||
66325 | GCC AAA AGA AAA GTG TAA GCA G | Mutant Forward | A | |||
66330 | GCT GTG AGC ACA AGT GGC TA | Common | A | |||
66331 | Fluorophore-1 | TCC ACA CAA GTA GCT GTG GC | Quencher-1 | WT Probe | ||
66332 | Fluorophore-2 | CCC ACA CAC TGC ACC TCC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66324 | 0.40 uM |
66325 | 0.40 uM |
66330 | 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 |