Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:163086742-163086845 104bp CAGCCACACGCAGAAAATG GCAACATAGTTTCCTTAGTTAGGC
Mut = 113 bp
Wt = 104 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ACAAGTCTTTCTCCGACTTCTAAGTGTACCCCCAATGTCACTCCTTACAAATAAACACATGATGTAAAAAAGATGTTTTTAAAAGACCACACAACCGAcgctgatgaataaggggtttgattatgctaatgaagcccgaaactgccaagggccgggtacgtgtcataactacatttgaacagcagctttcctcaagccacttcccccacccagtggacagtctgtccctgatgtcataagctgcccattgtgaccacctgttctgtcctttgacaacacagggtatcttagcgtcaaagcatcattataacctgctaacttcacctccttggaagctactgtttcgggctcttgtacctgcctgagttaacgtctctggtggctgttttctggctttctctgcatagcgattgctgacagaacgtgagtgagaatttcactccagagacccaagagccaaagcctggcagaagacagcacaatctttcacaggaagaggcttttcctttcgaggtgtaccacgtacactttgggcagcaggacacttggcttcaagtcaactcccggatggagccagaatccatagaaatttgtccttataaccctcaccaccgaatcccgctcagcaggttccagtaccacctggcgtcatgcaggaagaagaaccccaagaaagccaaaaagatggccagctgtaaatacaacgcctgccacgtggttcccatcagaaagctggctgaacatgaagctacctgtgtcaacaggagctccgtggaggaagaggacacattaggccctctgcaagtcagcctcccacagccgcagaaccaggacacactacaggttcgttggctttccaaccctgacatttggaatgttgacggcgccaactgtcacccaatgttcgtccttaagagttttgttccccaaaaacttgtttgtgaaagtgacatccaagagtcacggggaggagaccaatgcccagaagatcctcagactaggaccaggaaggcaaacttctagcaggaggaagagggctctcaaatgcgtggatggatcgcgcatctcatctgaataaaaacctgcaaagtaacggtccgggctgtgctggttgtttttaaaggtttagaaagagcggtggaggtggggtttagaactgagctgagaatgtaaattgaaccaccttccatggggaacacttaacagtcgctatgaaaatgctcactcaacgcacacaggctttgatcaagtcattcgacatctaaaacagcatcccgcacagccagccacacgcagaaaatggggttgagtaaaaagttactcccgtgttatttatagcagcactgaggtaccCATAGGGAAGGCCTAACTAAGGAAACTATGTTGCAACTTTACAAAGGAGAGA
This mutation is a 1255 bp deletion beginning at Chromosome 2 position 163,086,776 bp and ending after 163,088,030 bp (GRCm38/mm10). There is a 3 bp insertion (TTA) 5 bp after the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51525 | GCA ACA TAG TTT CCT TAG TTA GGC | Common | A | |||
51526 | CAG CCA CAC GCA GAA AAT G | Wild type Forward | A | |||
51527 | AGT GTA CCC CCA ATG TCA CTC | Mutant Forward | A | |||
51528 | Fluorophore-1 | ACT CCC GTG TTA TTT ATA GCA GCA | Quencher-1 | WT Probe | ||
51529 | Fluorophore-2 | AAA AGA CCA CAC AAC CGA CAT AGT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51525 | 0.40 uM |
51526 | 0.40 uM |
51527 | 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 |