Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:42970045+42970140 96bp TGCTTGTGTGTCTGTCTTTCTGT GGGAGGAAGATGATGTACACG
Mut= 95 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGACTAGCCTAAAACTCTCAATCCTCCTACTCAGACTTTATAGAGTACTGAGATTACAGACATGCAACACCATGCCATGTACACATTCCTAAATAGATATCCAATTGAGTAGAAATTAACCTCGTCCAAAGCCTTTCTTATTCTGTCAGCGTGGTCCTCTCTGGGGGTTAGAGACTGCTTGCTTCCCTCAGCCCAGGAGCCAGCATTTAGTGGCAGAGGgaagggactggggtggggcgaggatggaccacactgcccctaacttctcgtcccctttaggcctcaagaccggcggtggttcctcaggtggttccggtggctcctttcactacacctttcatggggaccctcatgccacctttgcttccttctttggtggctccaaccccttcgatatcttctttgccagcagccgctccactcggcccttcagtggctttgaccctgatgacatggatgtggatgaagatgaggacccatttggtgcctttggccgctttggcttcaatgggctgagtaggggtccaaggcgagccccagagccactgtacccccggcgcaaggtacaggacccccctgtggtgcacgagctgcgggtgtccctggaggagatctaccatggctccaccaagcggatgaagatcacaaggcggcgccttaaccctgacgggcgaactgtgcgcaccgaggacaaaatcctgcacattgtcatcaaacgaggctggaaagaaggtaccaagattaccttccccaaagagggcgacgccacacctgacaacatcccagccgacattgtcttcgtgctcaaagacaagcctcatgcacacttccgccgagatggcaccaacgtgctctacagtgccctcatcagcctcaaggaggtgtgtgtgtgtgtgtgtgtgtgtgttcgcacgtgtgtatgtatgtgcttgtgtgtctgtctttctgtctgttagagggtgataggaatcatttcctgcctgcctgccatggtCTTTTCCCGTGTACATCATCTTCCTCCCTCTCCACTCTTTGCCTACTTTTCCCTCACTCTGTGTTATTGTTTTCCAGGCGCTGTGTGGCTGCACCGTGAACATTCCCACCATTGATGGCCGAGTGATCCCTTTACCCTGCAATGATGTTATCAAGCCAGGCACCGTGAAAAGACTCCGTGGGGAGGGACTTCCCTTCCCCAAGGTGCCCACTCAGCGGGGAGACCTCATT
This mutation is a 775 bp deletion beginning at Chromosome 4 positive strand position 42,956,466 bp and ending after 42,957,240 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38657 | TGC TTG TGT GTC TGT CTT TCT GT | Wild type Forward | A | |||
38658 | GGG AGG AAG ATG ATG TAC ACG | Common | A | |||
38659 | GGT CCT CTC TGG GGG TTA GA | Mutant Forward | A | |||
38660 | Fluorophore-1 | CTG TTA GAG GGT GAT AGG AAT CAT TT | Quencher-1 | WT Probe | ||
38661 | Fluorophore-2 | TTT AGT GGC AGA GGC TTT TCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38657 | 0.40 uM |
38658 | 0.40 uM |
38659 | 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 |