Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:97324058-97324156 99bp CAGGCTCATACGTGTTTACTGT CTGATGGGATCACTGGAACA
Mut= 96 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCCCGCCAGTGTGCCAGGACCTACCTCCTATACACTTGCATAAGAGTTTGATATTAAGTGTACCTTCTTACATCTCAGCCGTTGTGTAGATGCGACTGGAATGACCTTTCCTCTCAAGGCCAGCTCCAGCATGAACTCTtccgaagggtttctgtttacacgcacagttgctttctcgtggtgtcagagtatgccagggaagggttgtagaatgaatgtattacagtggaattagactttgatgttgtcaggggagaggatgtggccccacgtgctgtttccagaggcctagtctacgtagtggtgcatgagagctcaatccattccagaggaggagccaccgtccagcgccttggccagcgtggtccagcacctcccattggagcttatggatggtgtggtccggaacctcagcaatgatgatagtgtgacagactcccagatgctgactgccattagcaggtgggtgccgagggtcagtgggtggagatggggctgtcagcggttagaggtcaggctcatacgtgtttactgtgagctcgacagtctcaccctgtttggccttgggATAGATATAAGTAACACCAAATCCTGTTCCAGTGATCCCATCAGACTAGGTACTAGCTGTGTGACCCTGGAAACGCTGCTGAACTCTCGGCCTCAGTCTTCCCCTATAAAAGTAGAAGTAGATGGCAACATGAGAAAGTAAAAACAGGG
This mutation is a 429 bp deletion beginning at Chromosome 7 position 97,324,102 bp and ending after 97,324,530 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44719 | CAG GCT CAT ACG TGT TTA CTG T | Wild type Forward | A | |||
44720 | CTG ATG GGA TCA CTG GAA CA | Common | A | |||
44721 | AGA TGC GAC TGG AAT GAC CT | Mutant Forward | A | |||
44722 | Fluorophore-1 | AGC TCG ACA GTC TCA CCC TG | Quencher-1 | WT Probe | ||
44723 | Fluorophore-2 | CCA GCT CCA GCA TGA ACT CTA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44719 | 0.40 uM |
44720 | 0.40 uM |
44721 | 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 |