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= 141 bp
Wild Type = 163 bp
>chr9:65999736+65999898 163bp AATTTGGCCACAAAGCCTCT GGAATGGTTCTGCTTCAAGG
Wt Sequence (deleted region in lower case)
ACTCCTAGATGAGAAAAAGAAACCCCTACAGAGATTTTGTGTGAAAAGAAAACCAGTTGTGCTGGCCTAGGGCCAGGCTGCACCTCTTTGGCCTTGCCTCTCAATCCGATTCTCCAGGGGGCAGCAGTTACCAACAACTCTTTTTGGCTTTAGCAGCGAGCCCAGGGAGTGCTAATGAAACTAGGCGGATGTACCAAGGTTTGTTAGCTTGCATTTGAGCTCGGTGAGGCTCCCTTTGAACCTTTTATGATGCTGCACTTTCAGGCATCCTTTGGGAGAATAGTTTACATCCTGAAGCAATAATGGATCAGAGACCATTTTCTTTTATACAGCAGTGGAAACGCCTTTGGCACTCTGGTATTAAATAACTGCTTATTTGGACACATTGTGCAATGGCTTTGCCTTTTCCGCccagtaatagttcccacagaagctattatactcagtttaacttttccaGGAgggactctgaaaggaaattgaaatcttagagattatactttatattacattcattgctaatgtcttatgttttagtttttgtgtctctcactctgtaccccatttgcccctctactttcaggtgaaggcctcccacaggtttattactttggaccatgtgggaagtacaatgccatggtgctggaactccttggccctagcttggaagacttgtttgacctctgtgaccgaacgtttactttgaagacggtgttaatgatagccatccagttggtaagttgatgtgtggttccagaagataaatggatcccttgcccacctccttgagggccctttaatgatgtattagctgcagtgagatctggtgacttcgtttttacaggcttaatggcattctagaaggatctagcagacttctgaaaggaactattgcagctgctgctttagctggttcctgtgtataatttggccacaaagcctcttggccagtatgtaatatgccaaacagctaaggtaggttcttagccacacaacttaaagggttacagagggttaactgagggAAGTCAATGCATCTGCAGTTTGCTCTATTTTAAGAGTTTGCACCTTGAAGCAGAACCATTCCTGTAACTGAAGTGGGGGAATATAATTTACATTAGGGAGAAAGAGGACGATTAGGTAGAGCTGTGTGAATTCAAGTAATTGCTTCAGTGTCTTATTTATAGGTCTTAGGCACACAGTTGTAAGCTTTTATCATGCCTTCTTTCTCTCACTGTTGAAAACCAGAAAAGACTGAAGGTACCAACCTTTATTCAAAGTCACGTAACTGAGATGTTACAGATAATATACCATATATAACATGTAGCATATTATATACTTGTGGGCTGACCAAGAAGCTTTACAGCTAAATGAAATACTAGTTCTACTT
611 bp deletion beginning at Chromosome 9 positive strand position 65,999,223 bp for 48 bp where there is an endogenous 3 bp retention (GGA) followed by 563 bp deletion ending after 65,999,836 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
36707 | CAG TGG AAA CGC CTT TGG | Mutant Forward | A | |||
36708 | GGA ATG GTT CTG CTT CAA GG | Common | A | |||
36709 | AAT TTG GCC ACA AAG CCT CT | Wild type Forward | A | |||
36711 | Fluorophore-1 | AGG TAG GTT CTT AGC CAC ACA ACT | Quencher-1 | WT Probe | ||
37947 | Fluorophore-2 | TGT GCA ATG GCT TTG CCT TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
36707 | 0.40 uM |
36708 | 0.40 uM |
36709 | 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 |