Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:45408742+45408832 91bp TCACTAGGCCCAGGAGACAC AACTCAAGACCCCAGCGACT
Mut = 99 bp
Wt = 91 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GACCTGTCCTGCCTCACGCTCCTGAGGGTGGAATGACAGGCTCATGTCACTAGGCCCAGGAGACACCACCTTTCCTGCCCctaggtctgaagaacagacattcagctatccaaaggcagtcgctggggtcttgagttgtgtagagcagtgaagtcacccctcaggaacactgacactgctattttcacattgaaacagtgactagaggttttggcctggctccctgacccctttctgtcccttctgcagttcctcgcccgactgaatggctccagtcccatgccaggagcccctccccgacagcccttcaacgatccattctttgtggtggagaccctgtgtatctgctggttctcctttgagctgctggtgcgtctggtggcctgccctagcaaagctgtgttcttcaagaatgtgatgaacctaattgacttcgtggccatcctgccttacttcgtggccctgggcacggagttagcccggcagcggggtgtgggccagccggctatgtccctggccatcctaagggtcatccgattggtgcgtgtcttccgcatcttcaagctctccaggcattcgaagggtctacagatcttgggtcagacactgcgggcttccatgcgtgagctaggtctcctcatcttcttcctcttcattggcgtggtcctcttttccagcgcagtctactttgctgaagtggaccgggtggacacccatttcaccagcatcccggagtccttttggtgggcagtggtcaccatgaccacggttggctatggggacatggcacccgtcaccgtgggtggcaagatcgtgggctctctgtgtgccattgcaggtgtgctcaccatctctctgcctgtgcctgtcattgtctctaactttagctacttttaccaccgggagacagagggcgaagaggcagggatgtacagccatgtggacacacagccctgcggtaccctggagggcaaggctaatggggggctggtggactctgaggtgcctgaactcctcccaccactctggccccctgcagggaaacacatggtgactgaggtgtgagggtcaactggggtctccagggagcagtggggtgggagggaggagggaaggcagggtcaggtgctgggttaaggactaagatggtaacaaaagggtacagattcttaatctgaaggcatgtcacatggtggctcctagggggaccttatgtgacaggaattgccaggatttgggttgtgtccagggctccccccATTGATTGCGCAATGTGGTAGAGCTGTGCAAATGTCCAGGGGCTCTATGGGTAGCACTGTAAGAGACTTGGCC
This mutation is a 1179 bp deletion beginning at Chromosome 7 position 45,408,776 bp and ending after 45,409,954 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51520 | TCA CTA GGC CCA GGA GAC AC | Common | A | |||
51521 | AAC TCA AGA CCC CAG CGA CT | Wild type Reverse | A | |||
51522 | CTC TTA CAG TGC TAC CCA TAG AGC | Mutant Reverse | A | |||
51523 | Fluorophore-1 | AGG TCT GAA GAA CAG ACA TTC AGC | Quencher-1 | WT Probe | ||
51524 | Fluorophore-2 | CCA TTG ATT GCG CAA TGT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51520 | 0.40 uM |
51521 | 0.40 uM |
51522 | 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 |