Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:96420476-96420613 138bp CCAGAGGTCACTGCAATAGG CCCACTCAATAGGCCAAAGT
Mut= 126 bp
Wt= 138 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
GTTAAGGATGCATACTAGGGGCATTGATGAGAAAATCAAATACATCAAAGAGAGAGGCTAGTGTTGAGTGTAAGGCCATTAAAGCCATTGGCTGTGATGTCTCACTCGCCATCATGTCCCCTCAGCTCTTTTTGAAAGTGGGTGATATCAGTCGGCAACACAGACACAACACTGCAAACTCACTCACGTTTGCTGTTTCAGACACAGTACACACACTTCTTGTTCCAGACATTTCCATCcaacttggatgacaaaagatgttctaggacagactgaaaggagtcagctgctgccttaccaatcctttatgacgtaaggtgacaagtgagcattttagagtcaatagctactgctaattttcaacttaggagcaccgtgagagcatccaggactcagcagtgagctctacactgacgttcaagcaggaccatagtctggagaccaggcaaatccgcacactgctctggaacctggcctatcggcagctgaaaaagaaagactggcagaggctggcccgcttgtggagttttacagaggaccagatcagagccattgaggaacagtggtcaggtgagtggcggccagatgacatggacaggatgaagccagttaaagttaggagtaatggtctttccctcctcaagagtcagtgcctggtgcacacaattggcagaagccagaggtcactgcaataggggtagttagcatggctcagtctgcaaaggtggtttggaatttagcagagagctacactcctgagaatGTTCGGTTAGCTGTGCTTGCAAAGGTTGACTACTTTGGCCTATTGAGTGGGGACCAAAGAAGCAAGGACACAATCCCTGGAATGCCTCTGGAATGCCAGTTTGGCTTTCACTAAAGTTTGCAGGTTGTTCTGGGATGAAAGGTCTTCCTCCGCCACAGAAAGCTTTAGTACCGAAGGTT
This mutation is a 524 bp deletion beginning at Chromosome 13 position 96420527 bp and ending after 96421050 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64405 | CCA GAG GTC ACT GCA ATA GG | Wild type Forward | A | |||
64406 | CCC ACT CAA TAG GCC AAA GT | Common | A | |||
64407 | CAC AAC ACT GCA AAC TCA CTC A | Mutant Forward | A | |||
64408 | Fluorophore-1 | CTC AGT CTG CAA AGG TGG TTT G | Quencher-1 | WT Probe | ||
64763 | Fluorophore-2 | ACA TTT CCA TCG TTC GGT TAG CTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64405 | 0.40 uM |
64406 | 0.40 uM |
64407 | 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 |