Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 130 bp
Wild Type = 133 bp
>chr1:181600444+181600576 133bp AATTCTCATGGTGCCGAGTG TGCTGAGTAGATGCCTGTTCTG
Wt Sequence (deleted region in lower case)
AATGAAATCACGGGTCTGGCGAGGTGTCCCAGCAGTTAGAACATGTACTGCTCTTGCTGAGGATCCCAGCAGCTGCATTGGGTGACTGACAGCCCCATGGAACTCCAGCTTGAGGGAATATCTGCTACCTCTGGTCTCCTTGGGCACCTGCACTGACGTGCGTATGTGCACACACACAATTTAGTAAAATAATTCTTTAAAACTACTAAAAAGTGCATTGGCCTCCAAGTCCTCTTTAATTCTCATGGTGCCGAGTGCTGACGGCCTCGGCCTCctctctataattgggtcagatatagttgtttaggagctgtttttgtaatagagacatcttgatggaaaggaagtcagaacaggcatctactcagcatattaacagattataaaggtttcatggttgaaagtcttcagtgtaaggttgccatcactgaccactataggcaggtgatctaggaggtctcacgttgacttggacttgaatgacgtgattctgttttttaacgttaggaagaccatcactatcatctgaagttggagtcataatctgtgatatcagtaatccagcctcacttgatgaaatggctaaacaggcaaagcttgtcctcaactgcgtaggaccggtaagtagtcggcctttctttgtatcagaacaaacagtccattcattcctaacaaaatctggtattttacagtccagagggacacacgggtggagggcagatggcagattgaaggccttccctccacagggaagtctttccattgatatgatagtcttccaaagtagtgacaggtctttgagaccctattcagagaatgtgctgattggcaccccttaccctgtgtccccCTCTCTAATGTAAAAATAAAATAAAAGTTTGATGAGCTAGAAAGATGGTTCAGTGGGTAAAGGCATTTGCCCTGCAACCCGACAAGTTTTGTTCAGTCTCTCAGACCCATACAGTGGAAGGGGAGAACTGGTCCCCAAAAGGTGTCCTGTGATCTGTACCCTTGCACCTTAGCATGGGTACATGCATGCATGTGTGTGTGTACACTCCCATACACAGTAAATACATGTCATTTGACATTTTTAAACGTTTGCATTTACAGTCCCAGCTTCTTTGCTTTACTCTTGTATCATTATGTAGAT
576 bp deletion beginning at Chromosome 1 positive strand position 179,670,350 bp and ending after 179,670,925 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38336 | AAT TCT CAT GGT GCC GAG TG | Common | A | |||
38337 | AAC AAA ACT TGT CGG GTT GC | Mutant Reverse | A | |||
38338 | TGC TGA GTA GAT GCC TGT TCT G | Wild type Reverse | A | |||
38339 | Fluorophore-1 | TGG TTC AGT GGG TAA AGG CA | Quencher-1 | MUT Probe | ||
38340 | Fluorophore-2 | AGA GAC ATC TTG ATG GAA AGG AAG T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38336 | 0.40 uM |
38337 | 0.40 uM |
38338 | 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 |