Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:133036136-133036234 99bp AGGCAAGCCTCTCTTTCTGTG AATGTGGAAAAGGATCCCTGA
Mut= 109 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTTCTGAAAGTGATTATGTTAAGAATCAGACACAGCCGTCTAGAAGATTGTATTAACACATCTTTGGTAGTTCAGAAGAAATTAGATCATCATGGTGTTGACAATAAGGGCTATTTTATGGGTAACATTGATAACTATTATAAGTCTGGAGTTTATcataggaattttaggaaatgtattcatagctctcgtgaacatcatagactgggttaaaagaggaaagatctctgcagtggataagacctatatggccctggccatctccaggactgcttttttattgtcactaatcacagggttcttggtatcattattggacccagctttattgggaatgagaacgatggtaaggctccttactatttcctggatggtgaccaatcatttcagtgtctggtttgcaacatgcctcagtatcttttattttctcaagatagctaatttctcaaattctattttccttgttctcaaatgggaagctaaaaaagtggtatcagtgacattggtggtatctgtgataatcttgatcatgaacattatagtcataaacaaattcactgacagacttcaagtaaacacactccagaactgtagtacaagtaacactttaaaagattatgggctctttttattcattagcactgggtttacactcaccccattcgctgtgtctttgacaatgtttcttctgctcatcttctccctgtggagacatctgaagaatatgtgtcacagtgccacaggctccagagatgtcagcacagtggcccacataaaaggcttgcaaactgtggtaaccttcctgttactatatactgcttttgttatgtcacttctttcagagtctttgaatattaacattcaacatacaaatcttctttctcattttttacggagtataggagtagcttttcccacaggccactcctgtgtactgattcttggaaacagtaagctgaggcaagcctctctttctgtgatattgtggctgaggtataagtacaaacatatagagaaTTGGGGCCCCTAAATCATATCAGGGATCCTTTTCCACATTCTAGAAAAAAATCAGTTAATAAGAACAGGAATTTAGGAAGGAATCTGAAATTATGAATCTCATAGGCCATGAACCTTCAGACAAAGGATTCATTAGAGAGATAGAGAGAGAACATTG
This mutation is a 861 bp bp deletion beginning at Chromosome 6 position 133,036,176 bp and ending after 133,037,036 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43578 | AGG CAA GCC TCT CTT TCT GTG | Wild type Forward | A | |||
43579 | AAT GTG GAA AAG GAT CCC TGA | Common | A | |||
43580 | CAT CAT GGT GTT GAC AAT AAG G | Mutant Forward | A | |||
43581 | Fluorophore-1 | TGT GGC TGA GGT ATA AGT ACA AAC ATA | Quencher-1 | WT Probe | ||
43582 | Fluorophore-2 | AAG TCT GGA GTT TAT TTG GGG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43578 | 0.40 uM |
43579 | 0.40 uM |
43580 | 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 |