Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:79966827+79966919 93bp TACCCAGGTCACTGGCATC TGAGCCCGTTAGGCTGACA
Mut= 92 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCAGCTTCTCACAGCCCAGTGCCCCGCCCCTTGATGGCATGGGGTGCACACAGTCAAACTTTCTGTATCCTGCCTCTACAAGTGGCCCTGACCCTGTGGCAAACTGGACATGGGACACCATTTTAACCCACATGGCCTTAGCCTCCcaagtagtggaaggacaccgcccccattgcccctctgtgtgctgagctggaggctcacacattttgagccagcaggggcctcttctggggacagggtagaatcccattattgtcagacactgaggacacagccagagctggccaagtgcacagtgatgagggcctgcagccttcctggttccatatgccaatggccctggagcaggtggacggggttcttatgggttggcatattctgtgttgcccctgaccttcagtgcccggcctctcacaggtctgcaccgggaacctcctggtcatcctcagccggcactaccaggacgtgtcctgcctgaagtttacaggggatggcagtcattttgtctcagcgggcaaggactgcttggcgctggcctggagcctctgcaggtagggcatgtgccagagatctacccagaaaggctatggaggtctcagggcaggcaataggctggtgactactgaggtatcccattcttgggggcttggggaggcaggccaggggctttccacgtggcctcactgctgttgtgtgcccagtgtgctacaggcagacccatccaggatccttgccccaagacatgtctggtcccagcacacgctcccaatcacagacctgcactgcggcttcggaggccccatggcccgggtggccaccgcttcattggaccagactgtgaaggtagcatccccagcctcgacccatgaattcctggcagccactggatctcagctaggagggagggagactggcatctcccttgccttggctgtttgggctccagacagcccataatccctatgcccgaccaaggacacccagagatggactgtccagcctgtctgctggaaattcccactcgtccacgcctcagtggccagatgcccttgagtgtgtagggttgtttgtttgttattggcacagtgttagagatggaacccaggccctgcatctgctagaccagtgctctacctgagcctcacctcagccctcttttatcttctagttttaaacagttttttgcttgttttttgagactgtctcattgtgtggccacagcttgcctggaacttggtttgtacaccagattggctagggattcacagacccccttgcctctctctgcctaggagcacagggattgagggcaggattggagacatattccctggctggggtgcctgtgattctgtgggggttcagaggctggtagcatgggcatgatggtgcgatggcccccatgcctcctggccccccgacttggctgtgtgcctggatgtccacagctccaggcttcccattcctgcagctctgggccatctcctcgggggacctgctgctgtctgtcttgtttgacatgggcatcacatctgtgaccatggacctggccgagcaccacatattctgtggaggcagcgatggctccatcttccaggtggacctgtgcagctgggtgagtggccctctgggactgagggagcccacagtcctttgccctacatcccaggcacttaacgacctgtctctacccccagcccggactgagggagcacagcttccagcctgagcagaacacagggaaggtcttcaaaggacacaggtagtcacctcaggaggtggggttagcacagtgcccacaccgtgaagcacctaacccccatcctcctctcccaggaaccaagtgacatgcctgtcagtgtcaacagacggcagtgttctgctctctggctcccacgatgaatcagtgaggctgtgggacgtgaagagcaagcagtgcttgcgcacagtcaccctcaaaggtgtgcccgaggcagcgtggccacacagggacaaggcagggccctgtcctgccagccaggcccctgatgttgtgttattgtgtccttcaggcccagtgaccaatgcagccatcattctggccccacccagcatgctgaaccctgaattcaggcccagcctgcccctgccccacttcaacaagcacctgctgggggctgagcatggagatgaagcccagggtgggggcctgcgcctgcagctagggctccacctgcaggtaggacccacagccactcttctagaacattctatactccagagttccttctgctgctgctggtgtgcctgtctggcatcaacagcttctgaacccttgctacccaggtcactggcatccactgctgctcttgccatccgtccccacaacatacgcctgtCTGAAGGCATACTGTGTCAGCCTAACGGGCTCAGCTGACTTGTCACATTGCTCTCAGTGAGTGGCCAGAGAGAGAGTCAGAGCAGCCTGAGGTCAGGAGACACCTCACCAGGAAGGGGCTTCTACCCAGTGTCCTCAGTGCTGCCCCTGGGGCTTTGACCTAGGTCTGTGCCACTCTACAGTGGCAGGCCCTTT
This mutation is a 2235 bp deletion beginning at Chromosome 10 position 79,964,652 bp and ending after 79,966,886 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44530 | TAC CCA GGT CAC TGG CAT C | Wild type Forward | A | |||
44531 | TGA GCC CGT TAG GCT GAC A | Common | A | |||
44532 | CTG ACC CTG TGG CAA ACT G | Mutant Forward | A | |||
44533 | Fluorophore-1 | CAT CCG TCC CCA CAA CAT A | Quencher-1 | WT Probe | ||
44534 | Fluorophore-2 | ACA TGG CCT TAG CCT CCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44530 | 0.40 uM |
44531 | 0.40 uM |
44532 | 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 |