Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:181683384+181683467 84bp AGAGAGGTGGGAGGTAGGTC AGTGGACAGCTGGGGAATG
Mut = 82 bp
Wt = 84 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGTCATAAGAGAGGTGGGAGGTAGGTCCCTGGGTCCCAAatagccattgggagagatgttttctgtccctgccattccccagctgtccactgtgggagccaagagtccttggtgaggatccgcctctggaacaggcccctccgcctctgactcaggccacttgtcttgcagtccacccgtgttgggatgtctgtcaatgctctgcgcaagcagagttcagatgaggagctcattgcacttgccaagtccctcatcaagtcctggaagaagctcttgggtgagtctcagaatgtaaagggaagctcttgctggtcagcacagcatagagtgctggctctgctaccctgattgaaactatatatggtgggatatgacaaacatagatcagaaaggactcatccctgggctggacatggatgaagatggtagaagatggatgtcatctgttgtgatggggaggtgatggcctgggatgtatgatcattgagacaagggtatATGCTGCAGAGGAGGTTTTCAGgctgcaggagGCCAACAGAAAAGGAGATCCAGGCAGGATGC
This mutation is a 459 bp deletion beginning at Chromosome 2 position 181,683,416 bp and ending after 181,683,874 bp (GRCm38/mm10). There is an additional 10 bp deletion (GCTGCAGGAG) 22 bp after the 459 bp deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49947 | AGA GAG GTG GGA GGT AGG TC | Common | A | |||
49948 | AGT GGA CAG CTG GGG AAT G | Wild type Reverse | A | |||
49949 | TCC TGC CTG GAT CTC CTT TTC | Mutant Reverse | A | |||
49950 | Fluorophore-1 | TTG GGA GAG ATG TTT TCT GTC CC | Quencher-1 | WT Probe | ||
49951 | Fluorophore-2 | TCC CAA ATG CTG CAG AGG AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49947 | 0.40 uM |
49948 | 0.40 uM |
49949 | 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 |