Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
>chr16:43943235+43943339 105bp AAACCAAAAGCACCATCAGC GCCAATCGGAATGTACAACTG
Mutant= 91 bp
Wild Type = 105 bp
Wt Sequence: GGCAAGGGGAAGCAGGAAGGTTAATTCCAGGACAGTCAGAGCTCTATGAAATGCTGTCTCCAAAAACCAAAAGCACCATCAGCCTTGGCCCTggcagcagccgtcacagctctgaccactctccccacttacacctgaaggtaatcccagttgtacattccgattggctcagcgtgccaaggctgatggtgtctgaggcactcaggcctggagaagggcagctctagctgaggtgcagcgtagctgtgccactgaacagaacaataccagtgagaccacagcaggagcttgtctcatcagtgtttgagttagaatgtcatcttttcccctaggtcactatcctgtcccctttgtgggaaaggccacctgtggagagcagtggttgtacactcagccattccccagaaagaaccaccaagggtaattgtcagggtcctctcccagatgtctctgggagcacacctggcacggagcagtgcaaggctgtagcagctgagacctctgagcagcctggtagctgtgaacgacctgggaccacaaggcaggaggcagaagctgcttgtgtgaggcatttccaccgtagccatgtgtaccagcaacagctgattgagaagcaaaagaagaaacttgagcagcagcagaaaaccattcagaagctaaaggaaaaccagcgcttggccgaggcccggtgggcagctaagcgagccccggcagtcacagaaggacagaactgccttcagtcagaccccagaggagcaagggacccgcagggcacctgccagaggcttctgaagtatgtcagcagggtcacagcactcggaatgtgtttgcagggttttcctctaagctcagtagctttgcttggcttcctgtatgctcacaaagccaggaacctcatgtatgctgactgtcccatgccaggtgtacagagataagcaaatggccgcctagacggataccattttacagtgagccctgggtggttgtgagacaaacactggaattatgtaggaaaagagaaaaatacgtgttctaagcttttgggtttctgatggtttaatgctgagaaacgagGGCAGACAGCATGGTCCCCAGGAGCAGCCCCAGGGCTGGCTCATTGACTCACTTGAATTTTCAGTTGTTCACAGTAAAAC
Deleted Region: ggcagcagccgtcacagctctgaccactctccccacttacacctgaaggtaatcccagttgtacattccgattggctcagcgtgccaaggctgatggtgtctgaggcactcaggcctggagaagggcagctctagctgaggtgcagcgtagctgtgccactgaacagaacaataccagtgagaccacagcaggagcttgtctcatcagtgtttgagttagaatgtcatcttttcccctaggtcactatcctgtcccctttgtgggaaaggccacctgtggagagcagtggttgtacactcagccattccccagaaagaaccaccaagggtaattgtcagggtcctctcccagatgtctctgggagcacacctggcacggagcagtgcaaggctgtagcagctgagacctctgagcagcctggtagctgtgaacgacctgggaccacaaggcaggaggcagaagctgcttgtgtgaggcatttccaccgtagccatgtgtaccagcaacagctgattgagaagcaaaagaagaaacttgagcagcagcagaaaaccattcagaagctaaaggaaaaccagcgcttggccgaggcccggtgggcagctaagcgagccccggcagtcacagaaggacagaactgccttcagtcagaccccagaggagcaagggacccgcagggcacctgccagaggcttctgaagtatgtcagcagggtcacagcactcggaatgtgtttgcagggttttcctctaagctcagtagctttgcttggcttcctgtatgctcacaaagccaggaacctcatgtatgctgactgtcccatgccaggtgtacagagataagcaaatggccgcctagacggataccattttacagtgagccctgggtggttgtgagacaaacactggaattatgtaggaaaagagaaaaatacgtgttctaagcttttgggtttctgatggtttaatgctgagaaacgag
982 bp deletion beginning at Chromosome 16 positive strand position 43,943,264 bp and ending after 43,944,245 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37043 | AAA CCA AAA GCA CCA TCA GC | Common | A | |||
37044 | GCC AAT CGG AAT GTA CAA CTG | Wild type Reverse | A | |||
37045 | GAA AAT TCA AGT GAG TCA ATG AGC | Mutant Reverse | A | |||
37049 | Fluorophore-1 | AGA CAG CAT GGT CCC CAG | Quencher-1 | MUT Probe | ||
37050 | Fluorophore-2 | AGC TCT GAC CAC TCT CCC C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37043 | 0.40 uM |
37044 | 0.40 uM |
37045 | 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 |