Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 113 bp
Wild Type = 110 bp
chr7:102098070+102098179 110bp GTGAAGACTGCCCACACCTC TCAAGGCCCTGCATTTCTAC
Wt Sequence (deletions in lower case):
CACAAACCCATGGCTGTCCGACTCCATTGCCTGTGTTATTTGTGTCTGTTTCGGATCCTGCCAATTgcatgcatcagctatgtcgcacagaaatgaaaacctgtccccagcagtccctggccagtgttttaaacttacactttggcctctgtggcacctcccttgtgtccataggttcgagctgttcctatcctgcccctgagcctggttccagatacctttgatgatgcctatgtgggctgctctgaggagatggaggagaaagcaggcctgctgctaaaggaagagatggcacgccatgccctgctgcgggaatcctgggaagcagcacaagaggcctgggcacaccggcgtcacaagctcactctacctcctggcttcaaagcccagcacggagtagcgattatggtgtacaccaactcatccaacaccttgtactgggagctgaaccaggccgtacggacaggaggtggctcccgggagctctacatgaggcacttccccttcaaggccctgcatttctacttgacccgggctctgcagctgctgcggggctctggagggtgcagtaggggacctggggaggtggtgttccgaggtgtgggcagtcttcactttgaacccaagaggctgggggactctgtccgattaggacagtttacctccagctctgtggatgagagagtggctcgcaggtttgggaatgccaccttcttcaatctaaggacttgttttggggctcctatccaggcgttgtctgtcttccctgaggagcgtgaggtgctgatacccccacacgaagtcttcttggtcactgggttctcccaggatggagcccagagcatagtgactctctggagttatgatcagacctgcagccactttaactgcgcctatttgggtggtgagcgatgaatgggagacagagaatggcagggtgggtgaaagaagccagtgtattctgacccagtccccagagccttcaggaaccacttggttagtgagattggggctccaccaggtcctgggagagttcaagatctgtggacatcctcagatgactgtcttctcaaaaggtcctctgtagtacctcgtAGACCAAGGAACAGCGAGAAGGAGGCAATATTGCTTCCGGTCTCAGTAGGCTCTGACTTGATGGGGCTA
This mutation is a 1029 bp deletion beginning at Chromosome 7 position 102,097,590 bp and ending after 102,098,618 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66920 | TCA AGG CCC TGC ATT TCT AC | Wild type Reverse | A | |||
66921 | GTG AAG ACT GCC CAC ACC TC | Wild type Forward | A | |||
66922 | CGA CTC CAT TGC CTG TGT TA | Mutant Reverse | A | |||
66923 | CCC ATC AAG TCA GAG CCT AC | Mutant Forward | A | |||
66924 | Fluorophore-1 | CAG CGA GAA GGA GGC AAT ATT GC | Quencher-1 | MUT Probe | ||
66925 | Fluorophore-2 | TAC TGC ACC CTC CAG AGC C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66920 | 0.40 uM |
66921 | 0.40 uM |
66922 | 0.40 uM |
66923 | 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 |