Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 71 bp
Wild Type = 74 bp
>chrX:120400478+120400551 74bp AGTTTTGGCACAGGACAATG TCTGGTCAAGAACAGTCACAAATAC
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion): AGTCAAAATATTTTCGGACTTGATGTCATTGAAACACCAGAAGGAGACAAGATGCCCCAACTGATTGTTCaaaaggagttagatagagaagagaaggatacctatgtgatgaaagtaaaagttgaagatggtggctttcctcaaagatccagtacagctattttgcaagtaagtgttgctgatacaaacgacaatcacccaatcttcatagaaaaggaaa... ...aggaactgcctcttgataataccttcgttggctgtgattccatctccaagtgctcctccagcagttctgatccctacagtgtttctgagtgtagctatccagtgacaactttcaaggcccctgtgTCTGTGCATATCAGACCGGTAGGTAATCCTAGTTTCTAAGTCATCCTTTTAACTTATTACTCTCATTCTTTTCATCTGATATAGAATTGCAATGAACATTGATTTCTAAGATGGAACAAAACAATCATATT
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49810 | TGT CAT TGA AAC ACC AGA AGG | Mutant Forward | A | |||
49811 | ACC TAC CGG TCT GAT ATG CAC | Mutant Reverse | A | |||
49812 | AGT TTT GGC ACA GGA CAA TG | Wild type Forward | A | |||
49813 | TCT GGT CAA GAA CAG TCA CAA ATA C | Wild type Reverse | A | |||
49814 | Fluorophore-1 | GAT GCC CCA ACT GAT TGT TCT C | Quencher-1 | MUT Probe | ||
49815 | Fluorophore-2 | CCA CCC TTA ATG TCC AAT GCC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49810 | 0.40 uM |
49811 | 0.40 uM |
49812 | 0.40 uM |
49813 | 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 |