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.
Mutant= 137 bp
Wild Type = 136 bp
>chr5:76859302+76859437 136bp GAATGTGGTCCCAGTTAAACG GCACAGCAGAGCTTGGAACT
Deleted Region: tggatgttaagagcgccgtgcctggcatgttggttagggaggtgtgggaaaaatgctaatcgcgcttcccttcccaatgccaggaccgacagaatgaaccgggcagctttgagagtcagtcctccctggaccagaacgggcagctaggagaagacaagcacatttggcacggagaggaaccagaacctttggaatcccacgaagagaaaagactgcacgaagagtattggcgggaacttgaagccaagtgcaaacgacaaaaagccgaagccgctgagaagaggcgccaggaagagcagaggcggcaggcgctggagaggaggctttgggaggagagcctcagacaggagctgctggaagaggaggaggagggggaggaggaagaagaggtgaaggaggagggggaggagggcgaagaggtgggcctgcagccacgggcagggaaggtacctccagaggaagggcaccagtctggtccggaggaacagcgctgtacagagcaagagctggggggagccgacgatcccgcccgcctggaggcagaagagcgcaggcgtgagcaggaagaggccgagaggcaggcggagaaactgaggcagcgcgaggctgagaggcaagaggaggaacagaagcagcgcgaggccgagaggcaagaggaggaagcgagacaatgcgaggccgagaggcaggcggagaaactgaggcagcgcgaggccgagaggcaggaggaggaagcaagacaacgcgaggccgagaggcaggcggagaaactgaggcagcgcgaggccgagaggcaggaggaggaactgaggcaacgggaggaggctgagaggcaggagaaactgaggcagcgcgatgctgaaagccaagaggaggaactaaggcagcgcgaggcggagaggcagcgcgctcaggaggaggacgccaaggggatgctgcaagaggaggaggaggaggccaagcgcatagaagagctgaaaggaaaggagacaccagagccgctggtggaggaaggtccgcagagtccagaaggagagagccagccttggctaacggacgatgctgaccagcgaagcccactgcagcgggaccttgagaagccaggggaacgggaacgagaagacctggaatccgcagggcagagagagatcgcggaggagcctagaggagagggcgagccggccgagcagagcggagacttggacgcgcactgcggtggtgtggacggggaagggaaagaaaccgctcagacagacagtccccagccgcaggagagacagatggaaggaacaccggccccggaggagaatgaagctactgcggcagacatagaccggaaggtggaggagctccggtggcaggaggtggacgagcggcagaccatgccccggccctacacattccaggtttcctctgggagtaggcagattctcttccccaaagtcaacctgagcccggtaacacccgctaaggacgcgagcttggccccagcggcccaggaaccgcccgcgccccgcggtgccgcctcacacgccctgccatcagcgctgagcatcccacacacggccattttggtcaccggagcgcagctctgcggcccggcggtcaacttgagccagatcaaggacacagcatgtaagtccctcctggggctgtcagaagaaaagcgccctatggacgtccccactgtggagtccagggccggcagtgggaagtccaggcctgccccagagtcaccaagcaatgcggccgcgctagccgagtgggcttccattcgatccagaatcctgaagaactcggagggcgaccaacgtggcgacagagagccggccagagccggggacgagccagtgcctcgagcgcggtgcgattcccgagggaacgtgcgcaggacgccgccagtcaacgccaagttctccatcatgcctgcctggcaaaagttttcggacagcggcgcagagaccttcagacaaagcctggacggggagagcggaaggaaaaagccggggctagcacccagcgaagaaacagcaccccagcctcatgcagcggctcagcaagaggtaagccaggagccaccagacaccacagacggatgcaaatttgccaaagaccttccatctttcctcgttccaggcctgccttctccgcagaaagctgcgtcccgcacggagtccacgaccactttggacagcgagaccacgagtgacgtgggaaacccagaccctgcgatgccaggaggcgaggaaaaggcctcgccctttggaataaaattgagaaggaccaactattccttgcgcttccactgtgaccaacaggccgaacagaagaagaaaaagaggcacagcagcacaggagacagtgtgggtggggccacccccgcaactgggagtgtctctggagagagcgagcctgaagccacgttcctcaagcatggcccatcccttccccaggagagaaagccagccctatcccctcggaaggactctgctgaaagccattctagcggtcactatgttgctgtggcccagtctggtctaccaccggccagtggccagaccccagctccagagcaagacagggcagtaagcaaaatgccatccatgcagaagccggccctggctcccaagcctgccagccagaccccaccttcatccccactctccaaactgagcagaccccacttagtggagcttcttgctaggcgagctgggaagctcgactccgagcccagtgagactgccaaggagagcagtgataaccagccaccctcaccatcactgcccgaggagctgaaggggcagaagagggatgagaaagatgtgccagaaaagaagcctgcttccccacctctgccagcgggccagcaagagagaccttcgctaattcctgaaacaggtagaaaaggtgtgtggctgggggttggatgtggcttcattcgatggggagggaattgagagggagcccacgggaaactggttctgggaacacctcagttaggaacaggtttgcccccaaatcagtccctttacattttatacattatttgaattggaatgtggtcccagttaaacgcagatattgttagccagtctgtagagatgatcaaaagtagaatttccttgaccagtgtctgcccttcagtctgggtag
3153 bp deletion beginning at Chromosome 5 positive strand position 76,856,248 bp and ending after 76,859,400 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37100 | TCC ATG GTG ATT CTA AGT GCA G | Mutant Forward | A | |||
37101 | GCA CAG CAG AGC TTG GAA CT | Common | A | |||
37102 | GAA TGT GGT CCC AGT TAA ACG | Wild type Forward | A | |||
37103 | Fluorophore-1 | TCT ATA CTC AGG ACC ACC TGC C | Quencher-1 | MUT Probe | ||
37104 | Fluorophore-2 | ACC AGT GTC TGC CCT TCA GT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37100 | 0.40 uM |
37101 | 0.40 uM |
37102 | 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 |