Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:52416793-52416912 120bp CTGCCTGGAGAGATTGTTGG GCCTTCCTAGAGCTCAAAAGATAC
Mutant= 137 bp
Wild Type = 120 bp
Wt sequence with deletion in lower case
TTCAGTAACTTAAGTCATTTGTAACTGTTGTGACTACATTTGAGGATCCTTTGCTGAGCATAGACTTCCTTAGGCCTAGAAAGAGAAGGTGCTTCTGACTGCCTGGAGAGATTGTTGGAAAGATGAGAAGTGGCCGTGTAGaatgagaagaggcaaagaaagtgagcttaacaacacagtccttgcttgatggagtatcttttgagctctaggaaggctaaagagtgttgtgtgctgacttataccacagaaacaaagaagaataaagtgtgggatctctgtgtgtggtaactctctgctggtttttatttcagagcacaaagttatcattgttgggctggacaatgctgggaagacgaccatcctttaccagttgtaagtattaactgcttaaagtttaattttgttgtttctagttattctctgtgcttttgtggtatttgatggatgaaatcaatgaaaagttatgtggatgcaagcagactaaccgtaagccagaagccagggactgtgtgctgagctagaaattcgcacgataccaggcatgctcattgttttgctttgatcctcttgtttttagaaaacaaggtatctgtttatacactttgcctgaatgatctgtaatattctaaaaccattatttatttgagctcttttatatctttagctgaatcattgtgtgagcgcaaaaacaagggcaattgggtctgagttagaagccatgttgacgtaccttgatgtaagcattggtgagatgaactagcttcctcactaattttgtaaacttttcccctcctagttccatgaatgaagtcgtacacacatctcctacaataggaagcaatgtggaagagattgtggttaataatacccgtttcctgatgtgggatattggtggccaagaatcacttcgtccttcctggaacacgtactatactaacacagaggtaacattcctaatgctagtgcttgcatgagctcctgagtcctggaacacgtactatactaacacagaggtaacattcctaatgctagtgcttgcatgagctcctgagtccttgagaagcattgggttcgagtcattacagtctttaaaaatggagcgtgagtacagtagtcttctattccagtcttgactcagttaacgttgagtctctgtagGGAGAGTTGGTGGGTGATCAGTCGGTTGTTTGTCCAGTAGTTACTCTTAAATGACTTGCATTTCATGATTTCTGATTGCATTTCCTTCTTGATGTTTATATGTGTCTGAGTGGGAGAAATCCCAGCCAAGTAACGGACTCTTATGTGGTTTGAGTCCTAACCAGTTTTTGCCGTGTATTGTCTTTTATGTAGTGTAGTTGCAGCTGAAAAATCTTTTAATCATAAAGCATTTTACACATAATCATATGGTTTTATTTACATGTTACATTC
A 1020 bp deletion beginning at Chromosome 2 position 52,415,850 bp and ending after 52,416,869 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45112 | CTG CCT GGA GAG ATT GTT GG | Common | A | |||
45113 | GCC TTC CTA GAG CTC AAA AGA TAC | Wild type Reverse | A | |||
45114 | CAT CAA GAA GGA AAT GCA ATC A | Mutant Reverse | A | |||
45115 | Fluorophore-1 | AGC TTA ACA ACA CAG TCC TTG CTT | Quencher-1 | WT Probe | ||
45116 | Fluorophore-2 | TCG GTT GTT TGT CCA GTA GTT ACT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45112 | 0.40 uM |
45113 | 0.40 uM |
45114 | 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 |