Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:103096477-103096601 125bp CAGTACACAAGGTGAAGTTCTTTTG GTGCCGGTATACTAGAATTGATAGA
Mut= 97 bp
Wt= 125 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (T insertion):
TGGCTCACAGCCATCTGTAATGGGATCTGATGCCCTCTTCTGGTGTGTCTGAAGAGATCAACAGTGAACTCACATAAATAAATAAATAAATAAATAAATAAATAAATAAATAAATCTTTAAAAAGTTTGCCATCATGCTGGCAGTTTCTATATGCATCTTGCCTCTTAATTACATTAACGGGCCATATCAAGTGACTGGCTATCCTATCTAGGTCTC^TTTCTGTAATGTACTGTATGTTATTCAAACAGTGACAAAAATATACTAACATCCTCAGATCGCATTGCCTCTgtatttactactaatacacaggcatgtgagcacaattttcaacagatatgtggctagttacatgtgaagtataacattttgaatcatttaagtttttgtgtttattttgtcctaagaccatgatacttagtatttttctgctgtactttttttgtttaccaagagttcgaaaccttgccctgtctagttattcactccatttaacaaccattgttttgcaggtgcttgattcaaaaggagttgatgtcacaccccgacctctctaccatccagatccacatgctgcgtcagcaaagccaaacaaactcttgacatcacaagaaggatcgcttggatcagattatatttcttcctatagcctctatcagaatacactaaaccctagcatgttagggcagtacacaaggtgaagttcttttgatttcactaattttgaacttccgtttggatTCATGCGTCCAGAATGTATATTAGTTTCCAGTTTTCTCACATCAATCTATCAATTCTAGTATACCGGCACTTACATTTTAATTATTCATGCTCAGAGCTTGATTAAATAGTTCCCATACATTCCAAAACAGGGTTTAAAGTTCACCTCTTGGTGAACAATTAATTGAAACCAGGAAAGTGAGAAGATGATAGAGTGAAGGATGCTCATTTGTTTACAATTTG
This mutation is a 450 bp deletion beginning at Chromosome 4 position 103,096,547 bp and ending after 103,096,996 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion 73 bp before the deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48158 | CAG TAC ACA AGG TGA AGT TCT TTT G | Wild type Forward | A | Wt F | ||
48159 | GTG CCG GTA TAC TAG AAT TGA TAG A | Common | A | Common | ||
48160 | CTA ACA TCC TCA GAT CGC AT | Mutant Forward | A | Mut F | ||
48161 | Fluorophore-1 | CAC TAA TTT TGA ACT TCC GTT TGG | Quencher-1 | WT Probe | ||
48162 | Fluorophore-2 | CCT CTT CAT GCG TCC AGA AT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48158 | 0.40 uM |
48159 | 0.40 uM |
48160 | 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 |