Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:72556493+72556589 97bp GCCCACGAAAGCTATTTACC GGGGACACTGAAGCAAAACA
Mut= 95 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAGCATCTACATCTTTGGGATTTTCCGAAACCCTTTCTATCCGAAGGATATACACGCTGTGTCTGTATTCTATGACAAGCAAAACAAGCTCCTGAAGATTGGTGCTGTCAGATGGATTTTGCTAACTCTAGGTAGGAAGGTTCTATTTATCAACTCTTACAAACTGTAGTTAGGGAAGACTGCTCACATTTATGAAATAGACATAATCTTTATGTATGATTTTTTTGTTGTTGTTTATTAGCTTTTCAGCTAATTGCTTAAATGTGCCCACGAAAGCTATTTACCAAGATAAActgtggtaggagcagtttctgcagtagtcagactcaccatgcattggtctgttttgcttcagtgtcccctttggcaatggtggccttccttgcgctggacagctccttacacgggctccactctgtgtctgtctccattggattcaccagggctttcagaatggtaattccagtgtgcactgtgaagcacggccggtgGCTGAGTGTAACTGTGGTTTTTATTAACAGTTTGCTTAGATTGCTGAATTCTACCTGAAAGTGGATCATTTGTATACTGTAGGTGTGGCAGAATACAGAAACTGCTTTACTGGAGACTGTGGTTGTATCAGCAGTGCACATGGTGTCCAGCACAGACTGCTGGTGGAACAGGAGCCTTGATACAGGAATCAGGCTGTTATTGGTAAGTGCATTTTTAACAGCTCTGTTGAGAGATTTTTTTTTTTTAACTCACAATCAAACTCATCCAATTGAAGGCTTCTT
This mutation is a 198 bp deletion beginning at Chromosome 12 position 72,556,521 bp and ending after 72,556,718 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47640 | GCC CAC GAA AGC TAT TTA CC | Common | A | |||
47641 | GGG GAC ACT GAA GCA AAA CA | Wild type Reverse | A | |||
47642 | GAT CCA CTT TCA GGT AGA ATT CAG | Mutant Reverse | A | |||
47643 | Fluorophore-1 | CAG TAG TCA GAC TCA CCA TGC AT | Quencher-1 | WT Probe | ||
47644 | Fluorophore-2 | AAA GCT GAG TGT AAC TGT GGT TTT TA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47640 | 0.40 uM |
47641 | 0.40 uM |
47642 | 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 |