Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:21936978+21937081 104bp TGTTCTTTGGTTGGAGGTTTG CCCACATTCATGGAGGACA
Mut= 108 bp
Wt= 104 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAAATACTCCTCTTGCCCATAGGCTGCACATAAGAAAGCAGGTTGGTTTGTAATGACTTCCCTGGGCCACCGGGTTCTAGTCATCTAAGAGTCCGTAAGACCCTCCCGCATGTCCACTATGCTTCTGGTCTCTAAACCCCGGATGAGGTAAGACCTATCACACACACTTTTGCTTGTTCATAGAGGGCaacaatcccacagggtcctcttgagaaccgaggcaacggaaaactaacaggtgggctatattctctgtttctgttttggttttgacaggaaacagcaagagctcgactccaaagatgctattattttgcatcagtttgcaagacctaacaatggtgtccccagcttgtctcctttctgtttgaaaatggaaacttacttaagaatggctgacctaccctatcaggtacttgtttgcaaatatgttttattcttgcgacatcgatatcacctagaggatggggtagttaatgtcacacaagtggatatcagagaaggcagggtctgaaggtaaccatacccagtcgtcagcaagagagacatgtgactccaggatctattgacttttgtttttccatcagttctaatgagggactcataatatttcttttaatgtgttctttggttggaggtttggctaggcttatggaacagctggtgagactgcctggtGGTACTTTTAGGTTTACATCTGTTATTTTGTCCTCCATGAATGTGGGGTTAGAAGGCATAATGGATTGGAAGTTCTAGGGCCTGAAGCTAGTGCCTCATGGCTCTCCCACCCAGCTATAGCTCTGGCCTTTGAAGAGATTTTAACTTCCCTATGTTCTGCTCAGGTGGTTTTTAAGGAAGTACCTG
This mutation is a 490 bp deletion beginning at Chromosome 4 position 21,936,545 bp and ending after 21,937,034 bp (GRCm38/mm10). In addition, there is a 7 bp insertion [TTTTAAG] at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44126 | TGT TCT TTG GTT GGA GGT TTG | Wild type Forward | A | |||
44127 | CCC ACA TTC ATG GAG GAC A | Common | A | |||
44128 | AAC CCC GGA TGA GGT AAG AC | Mutant Forward | A | |||
44129 | Fluorophore-1 | CTA GGC TTA TGG AAC AGC TGG T | Quencher-1 | WT Probe | ||
44141 | Fluorophore-2 | TGT TCA TAG AGG GCT TTT AAG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44126 | 0.40 uM |
44127 | 0.40 uM |
44128 | 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 |