Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 136 bp
Wild Type = 139 bp
>chr1:86024644+86024782 139bp AACAATGTCTCTGGTTTGGTG GTGAATGGCTAACCAATGTGT
Sequence (deletion in lower case, insertion in carrots)
GTAGAAAGCAAAACCTTCCAGCACCACATCTTTAAACCTTTAAAACGATAGGGTATTGAAGAGGCAAAGGAGGGACTGTCCATGGCCTACTGAATTTGTTAACTTTCTCATCACCGTGACCCAGTTATCTGAGAAATTACTGAAACGATGACTTATTCACGTTGGCTCACGGTTTTAAGAGTATCCACGTATCACGGCAAGGTGTAGCTCACAGTGAATCTGCTAGGAAGTGGAGAGAGGGATTATGTGAAGGGCCCAAGGCAAGATATAACCTGCAAAGATGTCCCACTGATGCCCTACAacacctgctagaatccgcctcttagcctttaccaactcccaataacatgacttctagaactgtcctagagactgtgtcccactcccaagaaccaggtgcagtcctcatctctctccttctctgtccagcagtgcctctgacctatgtggccccacgctcctgctcctgtgcagcctgcccgggcagctccgcttgctggcatcgtctggggctttgccatagccgaatctttgatgtcctcctacctcgggactggtcatccatgccagggagaggagtcccaaaccttctcaccttctacaggttccgcatgaacgggagggctaaaaagccttgggtgtagagttcccagggccaggctcggtcctcagatctggttggcaactgtgtgtaacttcctccaaacatcataggcttttcaaaagcaaagccgttccttctaaataacaatgtctctggtttggtgggccagctctttgtctgttcctttttctatgaaaccgtcttctctgctgacagcaccctcccaccCAGCCCATTGTAAACTCCAGAGCTGTGTGATTACACATTGGTTAGCCATTCACTTTTTTTTTTTTGACCCATACCATACACAGGGAGtgtctattta^GGTTTTTT^TTAAACGGCATGTTAATTATCCCTGGAACTCAATGATAAACCTGATCGCGTAACTCATAGGTCCCTTCTGCCAATAATAGCAAAATCTGTCCCACACAGAACATGCCATGCTTTTACCAGGAGTCCTCACAGGGGCCATGTTTTCTAGACCAGTCTATTAG
531 bp deletion beginning at Chromosome 1 positive strand position 86024199 bp and ending after 86024729 bp (GRCm38/mm10). In addition there is an indel (8bp insertion GGTTTTTT and a 10 bp deletion TGTCTATTTA) 85 bp after the 531 bp deletion
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37769 | TCT GCT AGG AAG TGG AGA GAG G | Mutant Forward | A | |||
37771 | GTG AAT GGC TAA CCA ATG TGT | Common | A | |||
37772 | AAC AAT GTC TCT GGT TTG GTG | Wild type Forward | A | |||
37773 | Fluorophore-1 | AAA GAT GTC CCA CTG ATG CC | Quencher-1 | MUT Probe | ||
37774 | Fluorophore-2 | CTG CTG ACA GCA CCC TCC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37769 | 0.40 uM |
37771 | 0.40 uM |
37772 | 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 |