Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:26008521-26008660 140bp CCAAACTCAGGAGCTCTTCATC CACACACCCCAAAAGATCATA
Mut= 145 bp
Wt= 140 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGTTCTTTGTTAGTTTTTGAGACAATAGCCTCACTGTGTAGACGAGGCTGACCTTGAATTCACAGAGATCTGCCTGCCTCTGCCTTCCAGCTCTGAGATTAAAGGTAAGGCCCAATATGCCCTTGTAGATTTGGAGGGCTATGTCTGGGATAGTGTTGCAGATTTAAgcaagtttgttggacattctgagaaataatctttttggtttgtttgatttttgagacaggtttttactgtagaattaaggttggcctcaagctcccagcagacttcttttctcagcctcgcaagagcagggattacaggtgtgcatcactgtaccgggcatgatctctttctttatcctactgcagttccagacagaactccggaagatcctcgtgtctctcattgaggtggcacagaagctgttagcactgaatcccgatgcagttgaattgtttaagaaggcaaatggtatgcgtgggtcctaggctgctcctaaaagtgtccccagccctcacactgaaagcaagttctgctttggttaatgggggagagggtttggtgtcagtaattgctaggtgaggtgagcctctactgatgaggctggggggcttcaaaggactctggaatcccctcatggcaggccttgtctttctcctcagctatgctggatgaagatgaggatgagcgagtggatgagactgccttgcggcagctcacagagatgggctttcctgagagcagagcctcaaaggcccttcggctgaaccagtatgcttgctttcttcttgccctgggcttccctgtgattctccttccaatctctggagtcatacttttttttttaatttttatttatctgtgtatatgtacatgtgtgtatgggtgcccatagaagccagagaggaagttggatcttctggagctagaattacagactgctgtgaactgcctgacacaggtgctaggaaccaaactcaggagctcttcatcttaatctaggagccagctccccgatcccattgagtcatttctaattgggtgttctagtagagGCACCTTATTGCTGTGCTTCAGAAGTGCTTTCATATATGATCTTTTGGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGAGGGTCTCTGTTACCTATGCTGGACTTGAACTCCTGGACTTATGTAGTCCTCCTGCCTTAGGCTCCTGAGTAGCTAGGATTGAAGGTGTGAACCATGATACC
This mutation is a 873 bp deletion beginning at Chromosome 2 position 26,008,577 bp and ending after 26,009,449 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46095 | CCA AAC TCA GGA GCT CTT CAT C | Wild type Forward | A | |||
46096 | CAC ACA CCC CAA AAG ATC ATA | Common | A | |||
46097 | TCT GCC TTC CAG CTC TGA | Mutant Forward | A | |||
46098 | Fluorophore-1 | CCG ATC CCA TTG AGT CAT TTC | Quencher-1 | WT Probe | ||
46099 | Fluorophore-2 | TGT TGC AGA TTT AAG CAC CTT ATT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46095 | 0.40 uM |
46096 | 0.40 uM |
46097 | 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 |