Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 96 bp
Wild Type = 129 bp
>chr6:83446426-83446554 129bp AGCAGGCCACAGAGGATATG CAAAGCCTGGATGGTTGG
Wt Sequence (deleted regions in lower case)
TGAAAGTTCTGTTATATTCTCAACAGTTACTCCATACTCTGGGATTGTTGGCTCATTAAAACTGCAGCCTAAATCCTAAGGTCACGTCTGCTCACTGTGCTGTGGTAACTGGTTAGCTGTCCAAGCCTTCACCCAGCCCACAGTGAGTTGTACAGCTGCCAAGTTTTGAAGCTGGAAAAGCTGGCCCTGGCTCCCATGAGATGGGCGGCCGTGGCagaagttactggctgcctccctcacctggatttgggaagcatcccaacatgtgtcatttgcattgcagctgtgggcaaatccacctttgtgaagttactcatgaaaactcacccggagtggcaggtggcgacagaacctatagcagagtggcagaatattcaggctgctggagcccaaaaagtaagttttcagctgtggggggtagttggcagacatgcacgcagtctaattggagtaatttaatttactctgaagacatgaagtagcccagcttgtctcctttttaaaataacttcattctagaaagcaggccacagaggatatgacttgtttggatatgtttagttttgatttttgtggcttctgtatgtctgtgCAAgcgcctgcctcctgtctttgtggtttCCCAAGCATGCCCAACCATCCAGGCTTTGAAAATGAATGTTGTCATTCCTACCCTTAAGTATTTATATTGTTGTTATTGAAGCTGGCTGGCAGGAGCAATAGTCAACGAGGACATCTTAGATGTGGGAAGGGACCTCACTGAGCATCTGGTCACCCTTTGAGTCAATGCA
377 bp deletion retention of 3 bp (CAA)of endogenous sequence and further deletion of 26 bp, beginning at Chromosome 6 negative strand position 83,496,866 bp and ending after 83,496,461 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38321 | TGT ACA GCT GCC AAG TTT TGA | Mutant Forward | A | |||
38322 | CAA AGC CTG GAT GGT TGG | Common | A | |||
38323 | AGC AGG CCA CAG AGG ATA TG | Wild type Forward | A | |||
38325 | Fluorophore-1 | CTG CCT CCT GTC TTT GTG GT | Quencher-1 | WT Probe | ||
39135 | Fluorophore-2 | AAA AGC TGG CCC TGG CTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38321 | 0.40 uM |
38322 | 0.40 uM |
38323 | 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 |