Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:62524396-62524487 92bp ATGCCTCCACCCACTCTGAC TGAGAGTCTTGCGCTTTCCT
Mutant= 109 bp
Wild Type = 92 bp
Wt Sequence deletions in lower case; insertions with carrots ^ct^ :
GCAGCTCCCGGACGGTGTCAACATCTCCAAGGAGGCCCGAAGCGCCATCTCCCGCGCCGCCAGTGTCTTCGTTTTGTATGCCACATCCTGGTGAGCTGAGCAGAGGCTGAAGGGAGGCGCAGCGGGATCCTGACCCCGCGTTCGTGGCCTGGAACCTACAGGCCCCAGTTCCCTGCCTCAGTAGAGAGCGACTTACGGCTCTCTTCCAGTTGTCACACATTCTGGAGCAGGTTAAACAAGACGGTTACCGTAGCCCTTTGTATGGGAAGGGGATGAACTGAGCCATGCCACACACACACACAATCCATTCATCTACGATAAATACGGCTTTCCCACCCCATTCCTGGGGGTCATGCCTCCACCCACTCTGACAGCTTCT^ct^gaagggcgttgctctttcttccagtgccaataacttcgcaatgaaaggaaagcgcaagactctcaatgccagtgacgtgctgtcagccatggaagagatggaattccagcgattcataacaccattgaaagaagctctagaaggtatccacgtactcttctgtctcttaaaccatatataaaaaaaatgctcatcagtatcagtgccattaagtcccgttcaataatacaggactggtcataactcccctctgtttagttggtaccatctcgggtatgtgataggggcctggttattccacctgtgcctcggtttgtctaattaaaccataatatcctgcttccttacagcgtacaggcgggagcagaaaggcaagaaggaggcttcggagcaaaagaagaaggacaaagacaaaaaggattccgaagagcaagacaaaagcagagaggaggaggaggaggatgaagagagactggacgaggacgaccagaacgaagaggaggaaatagacaactgaatggagagagtaggcagactgtggcgtggtggaggggaccctgagatcctgagacatgtctgtgtgaaggctttccctgcaaggcagagccatcttcggcactggtgcctgagatcaaaataaaagctgaccagaaagatcaaaaagaactcttcccaggctcagagcatctgagttacagaaaccggtcatgcgtcatcagtctgcaggttaggatgtttttgaataagcgattctgagtgactacatagtttggtattgcttgtctacttaaagtatatggctagctagtttctgatcccatttccctgcccctctgaataagtaataacatccatgttgcctgcagtgttccaagtcacagaagcactaaaggctcagggggaaacaatggggctttgagcctggtcacccagtttgactgtacagtagttgaccataggaactcgtgcttagaatggctgctctctaggagatgactaaggagctgtgagcgagcgaacgaggaaatgtcttcacctgatggtagcattacttacttggtgtctggctttctagacttacttagctttgtagtcctctagtgactgtagatgctcagtcgtctggggtggctgcactcataggacacacactctttaattctgagtgttgattagaatctgtcaaaaaaggtgccaattcttaaaagcaggggcttaatcttggaggtaactcagtgcctgccatggtaggtcctagtgctacctcctggaaggtataaaatcccagatccatctcccagacttatggctgctgcttttcattccctgggctcataacaagttccagagactttatttcttccaggcacagcctgttctgtctccatctctctggcttctgcttcccagcccggctttctcctcctcttacttatttcatcttctagtctagggactctgataacatatgtcatattccaaagtgcaaggtcctcagattccttagtcctaatctcctgccttctaaagagagttgagaacacctctgaaccttacggtaagaggaatggtgctcaggaatgtagcaccactgctgtggcttcgaacgtgtctgttggcctttctttttccccattgagtgctccaagcactttttcctgtcttagttgtgcacttgaggctgacaggtgatgtaatatagttggacactgtttactagtaatacagggaagagtttggtgagagtgactggagactgttgagaagattttaatgctttctacaaataaagaacattcttgtttaaaatgatgactcatacaatgtgcttttgCAAAGGACTCACCAAGACCCCCCTCTCTCTCCCCCTACCCTCACACATAGGCACACAGTCTTGTGTCTTGCATTACCAGCAGGTGAATGAGTATCACAAGTTAAGGGCACTGTTCTTAACCACAGCCAACATATCCTGAGATTCTCCTGGTTACTTTGCATATCTCACTAGCTCCCACTTCTGTTTGTTTGTTTGTTTTGGTTTTTTGAGACAGGGTTTCTCTCTGTAGCCCTGGCTGTCCTGGAACTCACTCTGTAGATCAGGCTGGCCTCGAATTCAGAAATTCACCTGCCTCTGCCTTCCAAATGCTGGGATTAAAGGTGTGTG
A 1834 bp deletion beginning at Chromosome 4 position 62,522,627 bp and ending after 62,524,460 bp (GRCm38/mm10). In addition, there is a 2 bp (CT) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44803 | ATG CCT CCA CCC ACT CTG AC | Common | A | |||
44804 | TGA GAG TCT TGC GCT TTC CT | Wild type Reverse | A | |||
44805 | GCT GGT AAT GCA AGA CAC AAG A | Mutant Reverse | A | |||
44806 | Fluorophore-1 | CCT ACC CTC ACA CAT AGG CAC | Quencher-1 | MUT Probe | ||
44807 | Fluorophore-2 | AGG GCG TTG CTC TTT CTT C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44803 | 0.40 uM |
44804 | 0.40 uM |
44805 | 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 |