Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:110652473-110652581 109bp GGCTCAGAAATCGTCTTTCC CTGACCAAGGTCCCAAACTC
Mut = 101 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ATAGACCATGCGATCTGGGAGGACGACGCAGTCACTCATTGTAGCTCAGGAAGCGAGTTGAGATCCGTATACTTGCCAGGACTGCGTGAATCCGTATACGTAGCCGTTTTCACTCACTCACACACACACACACACTCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACACACACACACACACACACACACACACACACACACACACACACACACACCCATGTTTTATTTGCGCCATTTCCGAGTTTGATACTGTCTCTGTCCTAGTGGGGAGACCCAGGTTCTGAGGCAAAGCGAGAGGAACTACATTTCAATACCCCCTTCCCAGTTTGCAGGGCTCAGAAATCGTCTTTCCCAAGCCCCGGtctggcatcttaaatagtttacagctgtgaagatgagaaacagccggaagattggggctgagtttgggaccttggtcaggcttggcacacataaatatgagtgtgagttagctctcctaactctgtccccagggtcccagggagccacatacaaatacaaggtgtggataagacatcgataccacagctgctgtaaccgcctagaggaactcctggcccatcccaccttccaggtcaaggtgagtcttgaagcccttagcccagcttttcctctcatctctaaAGTACTTCACAGCACAGGGGTAGAGGCCTTTGATGCGAGCATCTCTTCCTTCACTTGTCTGAGAGACTTCTACCTAGACATCTTTATTGCTGTTCTTTTCCACTGTGTGCTGTGAATCATCCGGACCTAAAGACATGGAGTCTGTAACCCTAGTTCCAGGCAGCAGATCTGCTTTGTACATTGTCACATTTGTTGAGGCACCTCCAAAACTGGGGGGGACAAGGGAATAGTTAGGTGGCCTCCCCTGCAGGCCCACCCTGTGTACATGCCTCAAGCCAAGTTCTGTTAAGAGTTCTATCCCAGGTCACCATCGGCACACACATGTAACATGGA
This mutation is a 283 bp deletion beginning at Chromosome 5 position 110,652,269 bp and ending after 110,652,551 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49260 | GGC TCA GAA ATC GTC TTT CC | Common | A | |||
49261 | CTG ACC AAG GTC CCA AAC TC | Wild type Reverse | A | Wt R | ||
49262 | AGA AGT CTC TCA GAC AAG TGA AGG | Mutant Reverse | A | Mut R | ||
49263 | Fluorophore-1 | AAC AGC CGG AAG ATT GGG G | Quencher-1 | WT Probe | ||
49264 | Fluorophore-2 | CCG GAG TAC TTC ACA GCA CAG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49260 | 0.40 uM |
49261 | 0.40 uM |
49262 | 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 |