Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 94 bp
Wild Type = 109 bp
>chr9:48393920+48394028 109bp CATTCTGTGTCTCCATGCACT CCATCTAGAATTTCAGGCCACT
Wt Sequence (deletions in lower case):
CATTCTGTGTCTCCATGCACTGCCTTGCACACAAACTCTGGgtccctgcctactatggaccaagaaatgaataaaagaaacttactaagtggcctgaaattctagatgggaaagttgtattatcactgagaactcacagaaggactagccaatctgggatctggagtgttgtctgagttgaagccactgtatattaatttctgtgcttgtctctttaaggtcaaatatagctgtggagattatggaaaaatccaatgcgattagtgtctccaaatgcaacagtaagtggtcctgtgctggtgggggatgagttacactgaacctatttaaaggtccttccctttctgttggacttggtcactgtctcagttcttgtaaggcgggccaaggcacaaagtccatgaatgagcaaatgagtgtgatcactagaactttggttaggtgctttagcagccacacctgtcagcaatctgctcatcaggctgaaggtcacctgtccatctaaGTCTTTACATAGTTCCTCATCTTCCCCTACCCTCTTTACATCCTTCCTTGGGA
This mutation is a 464 bp deletion beginning at Chromosome 9 position 48,393,961 bp and ending after 48,394,424 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66009 | CAT TCT GTG TCT CCA TGC ACT | Common | A | |||
66010 | CCA TCT AGA ATT TCA GGC CAC T | Wild type Reverse | A | |||
66011 | TCC CAA GGA AGG ATG TAA AGA G | Mutant Reverse | A | |||
66012 | Fluorophore-1 | CCT GCC TAC TAT GGA CCA AGA A | Quencher-1 | WT Probe | ||
66013 | Fluorophore-2 | CAC ACA AAC TCT GGG TCT TTA CAT AGT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66009 | 0.40 uM |
66010 | 0.40 uM |
66011 | 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 |