Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 96 bp
Wild Type = 115 bp
>chr1:167237034-167237148 115bp GACCAGCAAGAAGGGAGATG GACTCCCAACAACGAGAAGC
Wt Sequence (deleted region in lower case)
CATTGCATAGCCCTTGCTCCGGCTCTCGCTCCCACTGATGAGGCTGTGTAGGCGCTGAGTGCCCCGTGCTTACGTAGTCACTTTTTGCTCTGTCAGCATACCATCAGTTCCACTTGAAGATGGAGCGATACTTGGCATTCCGTTTTCATTCACCAGCTTGCAGAGAGGAGGCAGGGCCACTCTCCACACCTTACAGCATGGAGCTCTCAGAGCAGCAGGCGCTGAGGAGGAGGAAGGGACCCTGGGCTCCTTAGACCTTCCTTTCAGCAGGCTGGTCTTAGAGGGGTTTTATTTTTCAAGATCCCAAACCCATCCCCACCCCCACctcggcactgtcttaccgttttagttgactgtatattactagagctcagggaggcgccagcctcgagatgtgcacttcctgtgctcccacacacctctgactcctggctgtcgtcttctctcctcacagtcttccgtttgtgctaagatcgtccagcttttggggcagaatgaggtggattaccaccagaagcaggtggtgatcctgagccaggatagcttctaccgagtcctcacctcagagcagaaggccaaagccctcaagggccaattcaactttgatcacccgggtgagttgggagttgagagatgtgtgtgttgaaggtggatcgggtggtggctgtgtccacgaggcttagcttctcccagtctgttcctcctgtccacacataccataggggcctaatgaccagcttgcagggttatcatgtgcttagaggaaagaatgggttttgggatattttgcagagagctgattgaatcgaatgaatgccagccatgtaagcttgggtagagatgggtcagcccccggagcaaacacaccactggcctttctgattgatgtgtagcagcctgtaccatctccacccacccctcaacccccaccctgccccatgtggtctgcttggtgaccagcaagaagggagatgggagtcttaacgtggacccctacgtgaattctgctgcgagcttagatcagcacagtgctcCTTCAGGTCGTCTGGGCTTCTCGTTGTTGGGAGTCAGCCCTGCTTGGCATGGGACTGCAGTTCTTCCTAGTCCCGGTCTACTTTCCACTTAGCTTGAGCCAATCCCAGGGCCCCCAGCTGACCTTCGAGGAGAGATGGGAGGGGCCATGGCAGCACCAAGTGTAGGCTGTCAGCAGGTAACAAACAGCTCAGTTTGCCAGAACTCATTTAACACACCCTGTTTTAAGATCCAGGGAGTGTTTATATTCTGTCACGGGTGGACACTGGCTTTGGAGAGGCAACATACGGGGTGTGAAGTTTGTTCTGATTTTAGGACAGGTTA
709 bp deletion beginning at Chromosome 1 negative strand position 167,237,777 bp and ending after 167,237,069 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38042 | CAG CAG GCT GGT CTT AGA GG | Mutant Forward | A | |||
38043 | GAC TCC CAA CAA CGA GAA GC | Common | A | |||
38044 | GAC CAG CAA GAA GGG AGA TG | Wild type Forward | A | |||
38045 | Fluorophore-1 | AAG ATC CCA AAC CCA TCC C | Quencher-1 | MUT Probe | ||
38046 | Fluorophore-2 | ACG TGG ACC CCT ACG TGA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38042 | 0.40 uM |
38043 | 0.40 uM |
38044 | 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 |