Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 114 bp
Wild Type = 109 bp
chr7:4921736+4921844 109bp TCCAGTGCTTGCTCATTCTG CCTGGCTTTTCCCCAGTTC
Wt Sequence (deletions in lower case):
TAAGGAAGAGCCCCCACAGCATGGGCGACACAGGGAGGCTGCCCCTGTTCCTTGAAGAGCCGCTGTGAGTGGTGAGGAGGTCCAGTGCTTGCTCATTCTGATCAGAATCCTTTCACCAGGTGTGATGGCCGGCTCCCacgtggacatggcgccagcttccaccaccgagggaactggggaaaagccagggcccactgcgccagcacccaccccagctgctcagtatgagtgtggggaatgcggcaagtccttcaggtggtcatctcggctcctgcaccatcagcgcactcacacaggtgaacggccctataaatgcccggactgccccaaagccttcaagggttcctcagccctcctctaccaccagcggggccatacaggcgagcggccctatcagtgcccagactgccccaaagccttcaagcgctcttccttgctgcagatccaccgcagtgtgcacacaggcctgcgggccttcacctgcggccagtgtggtctggccttcaagtggtcatcccattaccaataccacttgagacagcataccggtgagcggccctacccgtgccctgactgccccaaggccttcaagaactcctccagcttgcggcgccaccggcatgtgcacacaggagaacgcccttacacctgtggcatttgtgggaagagctttactcagagcaccaatctgcggcagcaccagcgggtgcacacgggcgagcgccccttccgttgcccactctgccccaagaccttcacacactcctccaacctgctgctgcaccaccgcacccacggccctgcccccggccccgcccctgcccccgcccccccaggggagaccagcagagctgacaccaaggttctggtctctgatgcctacttgcagccccgtagcccgcctgagcctccagccccaccaccccagcccccacccgtggtgcccgagctttttttggctgctgctgagactacagtggagctagtgtaccgctgcgatggctgcgagcagggcttcagcagcgaggagctcctcctggagcaccagccatgcccggggccccctgtagccacccagtcccaggatgtgcctgccgagctgccccaggctgactccgccctaccacagcccccacctgccactccaggccctcctaactttgcttgtcttccttgtgggaagtccttccggacggtggctggcctgtcccgtcaccagcacagccatggtgcggccagtggacaggcctttcgctgtggcagctgtgatggtgccttcccacaactggccagcctcttggctcaccagcagtgtcacgtggaggaggcggcagccggacgcccacctccccaggctgaggttgcggaggtcacctgtccccaggaacctgtagccccagccactcctgccccaccccctcctcctccacctgccccggtggtttctgcagagcggccctacaaatgtgccgagtgtgggaaggccttcaagggttcttccgggctgcgctaccaccttcgggaccacacaggtgagcggccctaccagtgtggggagtgcggcaaagccttcaaacgctcctccctgctggccatccaccagcgtgtgcacacaggcctgcgagccttcacctgcggccagtgtggcctcaccttcaagtggtcgtcccactatcagtaccatctgcgacttcactcgggtgagcggccctatgcctgcactgagtgtggcaaagccttccgtaacacttcgtgcctgcggcgccaccggcatgtgcacaccggtgagcggccccactcatgtagtgtgtgtggcaagagcttcgcacagacgtccaacttgcggcagcaccagcgggtgcacacgggcgagcgccccttccgttgcccgctctgccccaagaccttcacacactcctccaacctgctgctgcaccagcgcactcattctgccgagcgcccttttgcctgccccatttgtggccgtggctttgttatggctgcctacctgcagcggcatctgaggacgcacaccccagccaccacaacttcgggcaccaccggctctgctgtagcatcgcagccccctgccccattggctgcagctccaactccactggctgctcaagatgttcacgtactccctaacctacaggccacactttcacttgaagtagctgggggaacagcccagcctacgcccccaggcccggctgctcccagttctcaaacatttctcctggtacagactgcccagggcctccagctgattccaagcagtgtccaatcccctactcctccacccccgcctccaccccctaaagtcattctgctgcctcctgccagtgctggaggtccgggcagtggtgctgcaaggccaggccctcggtctgttgggaaagctggacaggggacaggggtggtgtggtttccaggcccaggtggtctggggctgcaaggaggggccaatgcaggggctagcgggggagggcagagcctcattgttctgcagaatgtagggagtggggagacaggaccacaggaagtgagtggggtccagcttcagccagcacaggaagtggccacggtacaactacagcctgcacaggaagtgactacagtccagctacaaccagcacaggaggtgactacggtccagctccagcccctgacaggccaagtctccaattctaatggaggagctggcactacagaagctccaaacctgctgctggttcagagtggggcaactgaagagttgctgaccggccctgggcccggggaagtgggagacagtgaggccggtgctggtgtggtccaggacgtcctgtttgagacactgcagacagatgaggggctgcagagtgtgctggtgctgagtggggcggatggtgagcagactaggctctgcgtgcaggaggtggaaacgctttcccccggtctggcggagccagcggccactggcccatcaggacagaaactccttatcattcgtagcgccccagctactgatctgctagaaaatagcagcgttgcaggaggcaccacaacactgcaactcctggccccgtcagcacctggcccggtctctgcccctgtgggggtgcccgtagctcccccgtcccagatggtacaagtggtccccgcagtggctggaccaggggtcatggctccccagaacttgccctccatccagattgtgcagactcttcctgcagtccaactGGTACACACGTTTTGAAGAGACCCCCTCCCCGCAATTAACTGTTCTCCTCAGCTGGGCTGGGGTCAGCCCCAGAGTCAGGGGGGCTGCTGGCTGGACTTGCTAATAAAGACCCAGAATCTCTTCCTGGTTTTGTTTTCATGGGGGAGGTATAGTCATGGGCATAGCCCCTGCTTCAGACCTGGAAGTCTTCATTCTTGCTTCCCGGATCTGAGGTGACTTGCTTTGTACTTGGAGGGCTAAATGCTT
This is a 3088 bp deletion beginning at Chromosome 7 position 4,921,793 bp and ending after 4,924,880 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
60195 | TCC AGT GCT TGC TCA TTC TG | Common | A | |||
60196 | CCT GGC TTT TCC CCA GTT C | Wild type Reverse | A | |||
60197 | Fluorophore-1 | TGG ACA TGG CGC CAG C | Quencher-1 | WT Probe | ||
60198 | CCC AGC TGA GGA GAA CAG TTA | Mutant Reverse | A | |||
60199 | Fluorophore-2 | CCG GCT CCC GGT ACA CAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
60195 | 0.40 uM |
60196 | 0.40 uM |
60198 | 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 |