Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:100293641+100293723 83bp GAGCTTGCTGCTGAGGGCTC CCCTCGCCTCCAAACACAG
Mut= 96 bp
Wt= 83 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGCCCATGTGCTGCTCTTTGTACCCTCTCAGGTTACCTGGGCACGCTCACACACACACACACACACACACACACAAGCGCACACACTCACACACACATATACACACAACACACATGTATCCACTCATGCACACACATTCATGCATGCACATGTACACACACATGCACACACAAACACACACAATCTTTAACTAAAAGAATCCATTGTGTCCCAGGAGATGAGCTTGCTGCTGAGGGCTCTTTGagcaggtggtcaggtctcacatgagtgaaatgcccattgcctgtgtttggaggcgagggtcacagtctttctatttttacagctctgaaggatggctatgagaaggtagagcaagaccttcctgcctttgaggacctcgagggagcagcaagggccctgatgcggctgcaggatgtgtacatgctcaacgtgaagggcttggctcgaggcgtcttccagagggtcacgggctcctccatcaccgacctgtacagtcccaggcagctcttctctctcacagcggatgactgcttccaagttggcaaggtgacatgcaaagaagtccaactgctcgattctactttcttatggatggactgtgtctcagctgtctttaggtattccctttgcttgaggtaatgacAGGGTAGATCACTAAGACTGCATTGCAAAGTAGGAGGAGCTAAATTGAACCCCATCTTTTAGCTTCCCATGCTCTTGCCATAGGGATGAGAAGTCATCATATACTTAGGGCACAAGAAAAGCTTTTCTTTTCTTTCCAACAGGATATTGTGTATCCCAGGCTGGTGTTAAACTTGCCAGGGTCTTCCTGCCTCTACTTCTCCAGTGCTGGGATTACACCATGCCCATTTTTGCAGGGCTAGAAATCAAACCCAGGGTCTCATGTGT
This mutation is a 403 bp deletion beginning at Chromosome 7 position 100,293,665 bp and ending after 100,294,067 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47673 | GAG CTT GCT GCT GAG GGC TC | Common | A | |||
47674 | CCC TCG CCT CCA AAC ACA G | Wild type Reverse | A | |||
47675 | GCA TGG GAA GCT AAA AGA TGG | Mutant Reverse | A | |||
47676 | Fluorophore-1 | AGC AGG TGG TCA GGT CTC AC | Quencher-1 | WT Probe | ||
47677 | Fluorophore-2 | CTG CAT TGC AAA GTA GGA GGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47673 | 0.40 uM |
47674 | 0.40 uM |
47675 | 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 |