Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:99540354-99540473 120bp ATCTCAGGGAGCCCACAAC TCTCTTGGTTAGGTCTCTTCTCA
Mut= 117 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GACATTTAAACCTAATGGAAAGAGGGGATGGGAAAATGACTCAGCAATTAAGAGTGCCGATTGTTCTTCCAAAGGACCCAAGTTCAGTTTCCCGTACCCATCTCAGGGAGCCCACAACTGTCCATAACTgtagctccgtgtggcatacaggcacacttaaatgtaaatttttaaaatctttcttaaacagaagtaatgagaagagacctaaccaagagaatctgaacttttacccagtgtgccaaaaatcaaagtgcaatgcttttttcatcacattaacatcacagacacacacacacagagcactctgacattcttcactccatttccacccacaggaggtcaacctgcttcgtgagcagcttggcgatcggctcagcgtggagctggacactgcccccactgtggacctcaacaaggtcctggatgagatgcgatgtcaatacgagagggtgctggccaacaaccgcagggatgcagaggagtggttcgctgctcaggtcagtgccctcggtaaaggactggcttccgggagggtgccctgcctgagctctctaatggggcctttgtttcagacagaagagctgaaccagcagcagaagtccagcgcggagcagttggagggctgccagactgagatgttggagttgaagcgcaaggccaacactctggaaattgagctccaggcgcagcaaaccctggtgcgtgggatgaacagtgccagcctttgtcagcagcatccagagatgctatgctctttgacagaagtccagggagatgctcacagttcagcagcaggtctgggtatccaattagcctagctggcatttccctccctttcttcacatatgttacttgtccacagtagaaacagaatgacagatcaacgggctttgtcctctcaccagggtgcctccctggccctcctaagcctaagcatgtctccaataccatcagctagagacaagaaaagtctcagaacacagtgggacagggagagctgagccaaaggcccttaagcttagcaacagattttcctttcctctaatcgatgaacagttttacagattcaccgtagtgtctaaaacaaaaaaacaaacaaacaaacaaaaaaaaaccacatcaagtttcaagtggtcatacaacagctaagacccgcacatcaccatagccctccccacccacacaagaacaccatgcagtgaaagtcacgtataccaggtcccatcaggcactgtggtaccagttgagcttcatccaaggagtttacctagatgaacgtcacctgagcatccacctaggagaggcatggtgcaatctatggcagacaccttcactagcccgggtgaacaggtgggggaaagctagggtcaaaggaagaaagttccagaatgtgctgcagttctcagtagaaacagaatcctcctggtggttgaaccctgatcttctttgtaccaacactcactgcgcagatgctcagactcaagggtttcttcacaaatgatgacaaccctccttgagctacccaaaaaaagttctggctgacaattgttctaatgatagaagcgacacatttaaaggccttgagttgcttctaggcactgttccaaattctccccaagtatcagctcatctcacctccccagcctcccccatgacataggtactttgcattctccattgactctagaataaaacaggctcagagaagctaagtaactggccctgaggtctctgagctagaaaggggtacatcgagggtctgtcatagggtccttctgagaaggttctactcttgcagacggagtctctggagtgtactgtggcagaaaccgaggcccagtacagcacacaactggcccaaatgcagtgcctaattgacagtgtggagcaccagctggctgagatccgctgtgacctggagcgtcagaaccaggagtaccaggtgctgctggacaccaaggcccggctggagtgtgagatcaacacgtacaggggcctgctggagaaggaggacagcaggcaagttacacagacccccagagtccccccaggagaaccctgtttcctcaaggcctgactaaggccaattccatgtttcggggtcttccttttcaaacagtgaaagaaaggcgtggccagGTAGCTCAGTGGGTATGTCAGTTACTTCTTTGCTTCTGTAAACATGGCCAAAGACAACTTGAGGAAGAGAGAGTTTTGCAGATGGGTAAGAGTATGTCATGGCTGCAAGCATCAGGCCTGACAGCAGGAAGT
This mutation is a 2013 bp deletion beginning at Chromosome 11 position 99,538,431 bp and ending after 99,540,443 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43401 | ATC TCA GGG AGC CCA CAA C | Common | A | |||
43402 | TCT CTT GGT TAG GTC TCT TCT CA | Wild type Reverse | A | |||
43403 | ACC CAT CTG CAA AAC TCT CTC | Mutant Reverse | A | |||
43404 | Fluorophore-1 | AGC TCC GTG TGG CAT ACA G | Quencher-1 | WT Probe | ||
43405 | Fluorophore-2 | CTG TAG CTC AGT GGG TAT GTC AGT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43401 | 0.40 uM |
43402 | 0.40 uM |
43403 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |