Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:30599636+30599744 109bp AAAGGGATCCTCTCCTGTGC TTGCTCGCTTCACATCATTG
Mut = 107 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTCTCGACCAGTGCCTGCAACCTGGTCCCTTTAAGAGACCAGAACTGCGTCCGGCGTCGCAGCCCTAAAGGGATCCTCTCCTGTGCTCCTCCCCTGTCcgcgcggtgaatggttcgtgccggcctatattaacccgagatcttcctcccggacggcaatgatgtgaagcgagcaagtcggctaccgcgcgcacgaccacagagggcgcgctgcagatcgccgagaaccccgaagcggagccggacacccccggagggaagcaatgaggtagcggcagtccccggagcggacgatgtgcgcgtcctgccctccgaaccccgcgctaccttggccgagacggccgagcgcgagtctccggcgtcccctcggagcgaggatgcagtgagctgcgggaccgctgtgcttggcccgccgcggccagccagacctctcggaccacacttggggcaagctgcttattcagggaccagtccagccaaggggctcgccctcttctgcacccactgggggtggggagcaccggcccctgggcgcactgctgtagtttccgcggctcctggccctggcccaagacttgtgtgtgtgtcacgttcgctgaagaagagttccagcttatttctctcaccatcccggttgctttggccgagccgcccacctttatttatttgtacgcactcatttggcctttttctaaaatattttccgctccgctctcttattttgttgcttgtttgagacttttgtcctggtcctcctcatctaggcttctctctctctctctctctctctctctctctctctctctctttcctgaatatttttttctaatatcctaccccagtccctgcggggttcccaagcactatctggcctctcctgcctccatctccctgccccggaggtcctcccagcctgagttttatttttcgagcttgcccgggccaagcgtagcctgggttggtggccctgcggggctctcaccccgcgctcggccaccgccgccccagccactcccgggcgtcctccggagatgctgccgtggaagaagcacaagttcgagctgctggccgaggcgccgccgcggcaggcgtccaagcctaagggctacgctgtgagcctgcactactccgcgctcagctccttggcgagagcctgcccggaaggcgcgctcagccgggtgggcagcatgttccgctccaagcgcaagaagctgcacatcaccagcgaggaccccacttacactgtgctctacctggggaatgccaccaccatccaggcgcgcggggacggttgcactgacctagcggtgggcaagatctggagcaagagtgaggccggccgtcaagggactaagatgaagctgaccgtgagtgcgcaaggcatccgcatggtgcatgccgaggagcgagcgctgcgacgtcccggccacctctacctgctgcaccgcgtcacctactgtgtggcagacgcgcggctacccaaagtcttcgcctgggtataccggcatgagctgaagcacaaggcggtaatgctacgctgccacgcggtgctggtgtccaagccggagaaggctcaggccatggctctgctgctttaccagacatcagccaacgctctggcagagtttaaacgtctcaagcggcgggatgatgcgcgtcaccagcagcaggagttggtgggcgcacacaccatcccactagtgccgctgcgcaagctgctcttgcacggaccttgctgctacaagccgccagtggagcgcagccgcagcgctcccaagctgggctccatcaccgaggacctgcttggggaacagcaagaggaggagctgcaggaggaagaagaggagcatctggaggactgcctggaggaggaggaagaagaggacggtgtgggggatggggatccagcagaggaagaggctgaggcgcagagggcgctggtggtggccatgcagttggaatgtgaggacttgctggacccgctggaaaatggccacgaggaggcgttgggggacggcggggtctcgctgggccccagctcggggacatcacttcctctgtcagtctgtgcctcagacatgaaggcccaactgtcacagcttattagtgacctgggcgaccttagctttggcaatgacgtgagcaccctggagaccgatttaagggtgacacgcctgctgtcgggcgagagcacaggcagcgagagctccatcgagggcggaggcctggatgccacccctgtcagccctggcaacccatcgggccctgctgacagcaccagcctagatgagccctactcgggctaagctgccagaagacttctctgtgcgcccctcccaacatacacatagtggctccccaacagtagttctattccccctaacctccaggaaagggaaaatgatgcacttgaagcccagacccgactttggagttttctggggaagctattccctcttgctagttgccccagcgctttggctcagaggtggctcaggttgtgaacagggggatggggggcagggagggcgagaagcactacaaaggtgggtgagggagagctggctgaagttggaaaaggccaagggcgggctggaggtgcccagcagccggtttcctgtttgtgaggcagctggggctagggaaggaggggttaacttcctctccagaccccctgactggaaggcctctttctgtcatcaatatctcattaacaaagtcactttcaaaacgttggaggcttcaatgccactttacctgcccccagttctttcggataggggagcttgggctgtttacctcagactcaaactcttgaaagtttgccaaatttctcaaataactcttcttgggggggggggcagtgtatgaaagtcgtgttttgtttacagttaaagaaacctaatatctaaaaaacaaaaaaggaattttcctttagaaatacacaccttcctctggccccaaagctcctactccatgatgctatgtaaaatatttttgcactgttgtgaagtatttttgactttttgttttttgtacataactgtgttctcggagctgaatgtttatatctcttttgctgtgcaaaagatacctgtaaaatgttgtacggttgtatatatggaaatgtgtataaaacattttgttatttttttgagcagccttgtttcagttctgttggcatgtcagcagcggggtgagaataaagaggaaaagctgtcagaacaggcgagagcctctggaaatgctaccccagatgattggcggggaggaggtgggggcggggttgtgcctccagttttagtgtggtcctgctgtggctggaagagagccggaggtgagaccttgccacccatagcgtgcttttggatcagaccaaCTCGGGGTGTTTTATCTGTGGCCAGCCTCTGTATATTTCATATTCCCATTGTGTGTGTGTGTGCAGTTTTGCTTGCTTGTTGCTTTTGAGACAAGAGACGCACTGTGCGGCTCTAACTGGCCAGGAACTTCCTAATACAGACCAGGCTGGCTTCAAATTGAGAGATCTGCTTGCCTCTGTTTCCCAAATGTTGGGGG
This mutation is a 3280 bp deletion beginning at Chromosome 16 position 30,599,668 bp and ending after 30,602,947 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46436 | TTG CTC GCT TCA CAT CAT TG | Wild type Reverse | A | |||
48645 | AAA GGG ATC CTC TCC TGT GC | Common | A | |||
48646 | CAA GCA AAA CTG CAC ACA CA | Mutant Reverse | A | |||
48647 | Fluorophore-1 | CGC GGT GAA TGG TTC G | Quencher-1 | WT Probe | ||
48648 | Fluorophore-2 | CTG TCC TCG GGG TGT TTT ATC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46436 | 0.40 uM |
48645 | 0.40 uM |
48646 | 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 |