Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:105815164+105815302 139bp TCTGGACTCTGGACATTCTACA TTGTTCACACCTTGTCACTGAT
Mut= 134 bp
Wt= 139 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GATGTGAACTCATTTATGTTGAATGGTCTAGATGATGATAATGCTTTGGGCTTGTTTCTTAGAGTGTAGTCTGGACTCTGGACATTCTACAAAATTTAATTTCGAGTATAAGCAGAGCATAATGACGACCACCCCATAAGAatttggatagaaggacggcagcaagtgtgggtcgggggagccttgatcagtgacaaggtgtgaacaaggcagaggctttgggctgacaaggaacagtcgccagacttcaaagcttgtgatgaggctgacagcattgaggagctgtgtagagacttggctgtcagccctcctgatagtcacagatagggcagctttcatgcagagttctgtacacaccctgggctgccttcagggctgcatggatgtgggtagagtcatctcagcagccgcaggctcctcctgctatacccacttcctcgtttgcaaactgcttcacttctgagagaccagggccctttccagtagctttagggatggtaagttgtaagtgattcttgctaagtattccttttatgctcttaagtctcatcagagagataagaaatcagggcccagataaacaggtgcctgtgttacattcactgtaagtgagtctgcagtgaagctcacgtgaacatgctggaggcaaaggcaagttccagggttcaagttcagtaggattctaacagcaaacaatcacgccgctctgcttcttttgtttaaaacaaacaactagaaaatgcctccttattaggtaggtactgtacgctatgattgttaaatcgccatcctgaacatttcaactgtttgtgtccccatgcagctgtccatgacccaaacttggaggaggacaagtgaatgcatctcataaaggagaaggtaatataatcaatcgctttatctaacatttctacctgggcaaagatggaagtttaattttcaaggtacacggatactctctgttgacactctaccacacggcagaatgctctgctgatctcctctgccctgagttaatttgggacctttcctctcagagaaacagtaaacaatattaattttcacagcattgtgaagttggtgacgtcgctcaagcatgatgacagctggtcagtaggtcacaagttctcctcgttgttattcccacagactgggaagccaaggcggagaaggacatgtgagactgggcaggcttcagctgtcatctgtgggaagtggcttttggttgaccagcggtacttcccagtgtcagcacgtgtgttgtgtcggcagctcagaccttgcgctgaggggcccctgtcctcagccacccttcatcttgggaaaactctctggtgtagagcagggaggtagggaggcagggaggcagggaggcagggaggcagggaggcagggaggcttcctagtgctaactcaacttgtttaaaccattatactttatctaaataattgcattgaatacttcgatacaagtaagaacacgcaggagagaagtaggagaggctgccgtggaagaagaagaatatgcaggagagagaaggagagctgccatgtgctaaagggccaagagaacgtggcccagagggctgctcagttgggtcaagagcagccaagatggaacatagaaactagtaagtagtaactcagagttaacagtaggaaagtggattctaacagcatagagagtaggcagttacccagctatttgtgctgcctaaggcatatatatatatatatatatatatactttgatacaaagccctactatgtctgtgttgttatccctgttttaaaagaggactccatcgggttctcgtagttaggaagctatgaagtcaaactttaaaacccaagggttcctgactgcaaagtccactttctttccagttgccttgctgaagtgcccgctctgaatgaagaagaactgggtcctgcagtctgtccacaggaagaagactgtctgtgagcacagtctccatgttcactgtctcatgccgtccctgatatcatggaggaaatgaatggtcatttaagaatcctcattggctaaagagctggtggtttaaatgaatgaatctaatgcaaactgtgaagggctggtcctggtattaagaagatggacgattctgtgaagggaatgtgctaagacttgtttgttgaaaatggaattattaatggcttgtcagtaaagttaagaatgccttaaatcattcctctcttactctattcatagaagaaataaaacagatgaatttataaagggtttattgtctgccagacattattttttatatttatatgggtcatttaattctgcaatgatcctacttggaagatatagttatttattacagttgaaagttcgggggaagaacagaaaggttaagaaacttgcccaaggatacaaaggatggaaatggtaatggggcagcctgaacttccaaatctggacagctattcactgtcccttctgctcctgtctaatagaagggaagatcagacagaggtatgaaggtctgtgtctatatttagaaaaaaaattcttctttctcatttttttattcctagagggatagccactttttgcaatgctgagccatacTAATGGCAAGACAAGTCTACTGACTATCACTCTTAAGTCTCTCACCACATATCCTCATCCCTCTCTTTCCTTCTCAGCATGACTCTAGCATCCTCCTCTGCCCTTGGGAGTGAGGGGTGACTGTGGACTAA
This mutation is a 2461 bp deletion beginning at Chromosome 3 position 105,815,236 bp and ending after 105,817,696 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43201 | TCT GGA CTC TGG ACA TTC TAC A | Common | A | |||
43202 | TTG TTC ACA CCT TGT CAC TGA T | Wild type Reverse | A | |||
43203 | AGG GAT GAG GAT ATG TGG TGA | Mutant Reverse | A | |||
43204 | Fluorophore-1 | AAG GAC GGC AGC AAG TGT | Quencher-1 | WT Probe | ||
43205 | Fluorophore-2 | CCC ATA AGA TAA TGG CAA GAC AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43201 | 0.40 uM |
43202 | 0.40 uM |
43203 | 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 |