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.
>chr13:61002528-61002658 131bp CAGACATGGGGGTGCTTAG GCACTAGCGTTCTCACTACGG
Mutant= 131 bp
Wild Type = 145 bp
Wt Sequence: CTATAAAATTTTCTTATGGACAGAAAAAAATGTGAAGTGTCATTGAAAGCATTAGAATGGATTCTCAGTTCCAGATCTTTCACTTTATTTCACTTCCTGTAAtgacttacaacagtccagacatgggggtgcttagcagtgctgtggttctgattcatactcctcagaaggcaggtgtaactgagtctgttatctctttactgtttaaaggacggaccctgcaggtaccgtagtgagaacgctagtgctaatatcacagattatgtgaacctcccaccgaatgagctttacctatgggttgctgtagcatctattgggcctgtctctgctgccattgatgcttctcatgactctttcaggttctacaatggaggtaagcatatttccttctagatgtgaaaaaaatgaagaattgtgacatcccaatgccatgccatgctatgttcttatctataagattttatgtgagtttgagtttctcgattgccttccctgtgcaatcatactgagtcctatctttaaCTGATATGACCCTCTCATGAAAGTAAGTATATCATATGTTATAAAATTTATGTTTAGTTTATTTTTAAACTATCCC
Deleted Sequence:
tgacttacaacagtccagacatgggggtgcttagcagtgctgtggttctgattcatactcctcagaaggcaggtgtaactgagtctgttatctctttactgtttaaaggacggaccctgcaggtaccgtagtgagaacgctagtgctaatatcacagattatgtgaacctcccaccgaatgagctttacctatgggttgctgtagcatctattgggcctgtctctgctgccattgatgcttctcatgactctttcaggttctacaatggaggtaagcatatttccttctagatgtgaaaaaaatgaagaattgtgacatcccaatgccatgccatgctatgttcttatctataagattttatgtgagtttgagtttctcgattgccttccctgtgcaatcatactgagtcctatctttaa
420 bp deletion beginning at Chromosome 13 negative strand position 61,002,673 bp and ending after 61,002,254 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37053 | GGA TTC TCA GTT CCA GAT CTT TCA | Mutant Forward | A | |||
37054 | AAA ATC TAA AGC ATC CAC CAA A | Mutant Reverse | A | |||
37055 | CAG ACA TGG GGG TGC TTA G | Wild type Forward | A | |||
37056 | GCA CTA GCG TTC TCA CTA CGG | Wild type Reverse | A | |||
37057 | Fluorophore-1 | TCC TGT AAC TGA TAT GAC CCT CTC AT | Quencher-1 | MUT Probe | ||
37061 | Fluorophore-2 | CTG TGG TTC TGA TTC ATA CTC CTC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37053 | 0.40 uM |
37054 | 0.40 uM |
37055 | 0.40 uM |
37056 | 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 |