Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:81895045+81895152 108bp TGCAAACAAGCAGAACTTGG GAAGTAGCTTTGGGACTCAGC
Mut= 96 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCTTCAGCTTGCTACATAGCCAAGGATAATTTTAATTCCTGATCCCTGAGCCCTGCCTCCCATAATGCTGACATTCACTTGTTCTTCAAGTAAACTGCAAACAAGCAGAACTTGGGATCCTGCACACAGATGCTTCTGTGCCTGCTCACTGCCACACAtgtgagagggaaaagctgtgccaggctgagtcccaaagctacttccttctttgtgcccgtaggttgaaccggcctcttactctctcagagaagattgtatatggacacctggatgacccagccaaccaggagatcgagcggggaaagacatacctgcgtttacggcccgaccgggtggccatgcaggatgccacagcccagatggctatgctacagttcattagcagtgggctgcctaaggtggctgtaccatcaaccatccactgtgatcatctgattgaggcccaggttgggggtgagaaagacctgcgcagggccaaggtgagcccaagagggctggtggctgggtgtggagtgggggaagggagacagagcccaggctgcagaaggctgaggcactactgctgagctgcctgtcAGTCTTTCCATGGCACTGATGGTCTGAGGATGGAATCCAGACCTCATATGTGACAAGCAAGTGTTCTACCACTGAGCCATTCCCCAGCCCTGAGGGTTAACATCTTTTTAAGACTTGCTTTTAATTCTTCCTAATACTTAAAATGAAAATCTGATGCAAA
This mutation is a 418 bp deletion beginning at Chromosome 15 position 81,895,108 bp and ending after 81,895,525 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53935 | TGC AAA CAA GCA GAA CTT GG | Common | A | |||
53936 | GAA GTA GCT TTG GGA CTC AGC | Wild type Reverse | A | |||
53937 | CCA TCC TCA GAC CAT CAG TG | Mutant Reverse | A | |||
53938 | Fluorophore-1 | AGA GGG AAA AGC TGT GCC AG | Quencher-1 | WT Probe | ||
53939 | Fluorophore-2 | CTG CCA CAC AAG TCT TTC CAT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53935 | 0.40 uM |
53936 | 0.40 uM |
53937 | 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 |