Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:85461354+85461461 108bp AAGTCCTGGAAGTGCCTGAG GTTGATCCAACTACTGCTAAGATGT
Mut = 87 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTTCCTTGGTCTACCTTCAGTAGCTGTTAAGCATTGCTCTGTCCTTATTTGGCAGGCCAGCGCCAAGTCCTGGAAGTGCCTGAGAGtttcccaagtggcagacctttacattttcttaagcagcctcaggcccaggagatgtgcttaacatcttagcagtagttggatcaactgttccatgtttagcatgagactcattcaacttgtgtttttggcaggctgttaacaaaatcaaagagatagctgtgactgtgaagaagcaagataaagtgtaagtttccagggggctgtgtcagaaccttgatatcttgaggccaaatagggtattttggccaaataagcatgttgcagactttgtataagcgggtgggtggccttttggttcttgacagaccctttgatgtaaaacatgaaagagGCTGTAGACTGTCATTAAGAGCCACCGTGGGGGTGGGGGTGGGGTGACTGTATCTGCTG
This mutation is a 332 bp deletion beginning at Chromosome 6 position 85,461,376 bp and ending after 85,461,707 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50305 | AAG TCC TGG AAG TGC CTG AG | Common | A | |||
50306 | GTT GAT CCA ACT ACT GCT AAG ATG T | Wild type Reverse | A | |||
50307 | GAC CGA CAG CAG ATA CAG TCA | Mutant Reverse | A | |||
50308 | Fluorophore-1 | TTA AGC AGC CTC AGG CCC AG | Quencher-1 | WT Probe | ||
50309 | Fluorophore-2 | TGT CAT TAA GAG CCA CCG TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50305 | 0.40 uM |
50306 | 0.40 uM |
50307 | 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 |