Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:152873024+152873123 100bp GGCTGAATAAGTGGCTATTTTAGGA TCCTTTTCTGTCTCTTTGTGG
Mut = 103 bp
Wt = 100 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TCCCAAGTGCTGGGAGTAATGGTAGGCTGAATAAGTGGCTATTTTAGGATACTTCTGCAGTAGTctgactaggtttttggtctcttgtagttgacaacacttaccacaaagagacagaaaaggaaaatcttcagaaacagtctatcccatcaaatgattgttcttccctggatgctaagagagctgtatcaggaaatactcctgtccagcctcagaggtaagagtggggcacaaaagtggatattactggttctgctcaatcaatatagtaactccacagattccttatgttacttcagtgtttatggattgtagctTGCGGGTTTAATTTATAGGTAAGTGCTTATAGACACTTAGTTGCTTGGTTAAGGCACATTCATTTCAAAGCCAAGATTCAAACTTGTCGGTCTGACTTTAAAAGGTGTGTATGGATACAATTGTTTTTCTGATGTCAGTTCTCTTTCTATCTCTTTTTAA
This mutation is a 253 bp deletion beginning at Chromosome 2 position 152,873,064 bp and ending after 152,873,316 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50320 | GGC TGA ATA AGT GGC TAT TTT AGG A | Common | A | |||
50321 | TCC TTT TCT GTC TCT TTG TGG | Wild type Reverse | A | |||
50322 | AAT GTG CCT TAA CCA AGC AA | Mutant Reverse | A | |||
50323 | Fluorophore-1 | CTA GGT TTT TGG TCT CTT GTA GTT GAC AAC | Quencher-1 | WT Probe | ||
50324 | Fluorophore-2 | ATG CGG GTT TAA TTT ATA GGT AAG TGC T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50320 | 0.40 uM |
50321 | 0.40 uM |
50322 | 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 |