Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:143764495-143764605 111bp CAGCTCAGGAAGGACCCACT GGCCACTCTCTACACTTGGTG
Mut= 116 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GACTCTGGAAGCTGCAGGCTCTTGCTTTCCACACTCGGGGCACAGCAGCTTCGTAGTGAACACTGTCACCCTTTGCAGGGGCCTCCCAGAGCACCCACGGAAGCCAGCCAGGATGTTCTCCATattcaacatctggggcacattcaacaaagtgctcttcttcctcagcctcactgtcagccttgcagggctggtgggcaacgctctgctgctctggcacctcggtctgcacattaagaagggacccttcaacacctacctgctgcacctggctgctgccgatttcctcttcctctcatgccaagttggcttctctattgctacgatcgtgtcgggccatgaggacacactctacttcccagtcaccttcctgtggtttgccgtggggctctggctgctggctgccttcagtgtggactgttgcctcgcctacatgttcccctccttttgcagccccaaccgccgtcccaggttcacttcagttgtcctctgcctcgtgatctgggccctgaccatgccggccgtgctgttgccagccaacgcctgtgggctgctaaaaaacggaatgagtctgctggtctgcctcaagtaccattggaccagtgtcacctggctagcggtactgtctggcatggcatgtggggcaagcaagtttctcttaatctttgggaactgctgctcttcacagccaccccccaagttctgcaagttggcccagtgctctgggatccttctgttcttctgtcgcctacccctggttgtctactggtgcctgcggccagtcttaaaattcctgctccccttcttctttccactggccaccctcctggcctgcattgacagcagtgcaaagcccctcttgtactacatgaagggcaggcagctcaggaaggacccactgcaggtagccctaaacagggctctaggggaagagtcccagtcAGGTCTTGGGGGGCTATCCTTGCCCATGCACCAAGTGTAGAGAGTGGCCATTATACGCCCACCTACCCTGCCCTCAGAGCTGCCAGGGGGTCCCAGTCTTGCTCAAACCTTTGCG
This mutation is a 819 bp deletion beginning at Chromosome 7 position 143,764,544 bp and ending after 143,765,362 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42883 | CAG CTC AGG AAG GAC CCA CT | Wild type Forward | A | |||
42884 | GGC CAC TCT CTA CAC TTG GTG | Common | A | |||
42885 | TGA ACA CTG TCA CCC TTT GC | Mutant Forward | A | |||
42886 | Fluorophore-1 | CAG GGC TCT AGG GGA AGA GT | Quencher-1 | WT Probe | ||
42887 | Fluorophore-2 | CCA GGA TGT TCT CCA TAG GTC TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42883 | 0.40 uM |
42884 | 0.40 uM |
42885 | 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 |