Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:106085385+106085536 152bp CATCTGTGGTGAGTGCCAGT GGTCATCCCTTCCACTTTCA
Mut= 165 bp
Wt= 152 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGCCCCGTAAGATTTCTTTCATCCACACTGGTATGTCAACTGGTATTGTCGTTATGCAGGTCTTGTTTAGGTTCCATGGATGCAGCTATAAAAATTTTACCCACTCATATTCAATGTTCCCTGAGCTTTAGGTATAGGATTTATGTTACAGATGCATCAGTTGTGGATGAGGACCTCATGgtactcattttgaccagttgttgatttctgtaattgtccccatctgctgcagaaaagaaaacgtctttgatgagaagtgagagctacacctagcagggaaggccagcatgtgaactaagtgtctggagctaggcattggaatgtgaaatggctcaggaggacttggttcttgataattgatagtcttaattcctatgtcatacagctgagttaaacttctgaaatttctttcccaaaggtttcactagaagaaaagagtgcaaccattatttatgaccctaaacttcagactccaaagaccctccaagaagctatcgatgacatgggctttgatgctcttctccacaatgctaaccccctccctgtcttaaccaataccgtgttcctgactgttactgctccactgactctgccttgggaccatatccaaagtacattgctcaagaccaagggtgtgactggtgttaagatttcccctcagcaaagaagtgcagtagtcaccataattccatctgtggtgagtgccagtcagattgtggagctggtcccagacctcagtttagacatgggaactcaggagaaaaagtcaggagcttgtgaagagcacagcacgcctcaggcaggggaagtcatgctgaagatgaaagtggaagggatgacctgccattcatgtactagcaccattgaaggaaaagttggaaagctgcaaggtgttcagcgcattaaaggtaattttttttttgctcattttataaagctattctttgttttgatctttaaactttctgctttcttcccaagtatgagtttcagatccgtgggcctagaattgcttcctgcttcattgaagttagaagtgtagcatagtcccaggattaatgtcagtaaagttaatgattataatgaaaggttatgagagttgaacttcagtttagcaggaaaggcactgtgGTAGTGATTTGTGGAGTTTAGGCTTCCTGAACAGGCTGCCTTAGGCATATTTTATCTCTAATATATTTGTACTGGAGTGATGCAGAAACATGGAGACCATAGGAGCAGTGGTCATGTTGCTTGTTTCCAAAGTATGTCAATC
This mutation is a 951 bp deletion beginning at Chromosome X position 106,084,876 bp and ending after 106,085,826 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42276 | CAT CTG TGG TGA GTG CCA GT | Wild type Forward | A | |||
42277 | GGT CAT CCC TTC CAC TTT CA | Wild type Reverse | A | |||
42278 | TTG TCG TTA TGC AGG TCT TG | Mutant Forward | A | |||
42279 | TCA GGA AGC CTA AAC TCC ACA | Mutant Reverse | A | |||
42280 | Fluorophore-1 | AGA TTG TGG AGC TGG TCC C | Quencher-1 | WT Probe | ||
42281 | Fluorophore-2 | TGA GGA CCT CAT GGT AGT GAT TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42276 | 0.40 uM |
42277 | 0.40 uM |
42278 | 0.40 uM |
42279 | 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 |