Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:83315224-83315340 117bp AGTCACGCAAACGCACCA GGATCCCCTGCCAAACTAC
Mut = 111 bp
Wt = 117 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TGAGCCAGGTGAGTAGCTGCAGAATGTCAGGACAACTTACCTAATCCTCTCTGTCTCAAGTTTTCctgctgggaaagcagggctggcttgagaacaaaggctgtgtggtgcaggtggatccactgcggccctaaagtgtgtgtgcccgtgagcctagtggccagcatagttgtaacaactgtctcgtcctcaatgtctcctcaggctcgcggctctgcaggcggaaggaggaggcatgatgccgcctctcaggaagaccacgacaaaccctacgtctgcgacagtaagtatctccttgtactgtcatagcccaggctttctgtgccactttggcagtcacgcaaacgcaccagctctggtccacgccgggctttgctgtttccagctcccggtggtcacattccctcCTGCTGTGGCGAGGCTCTGGGCATAGTAGTTTGGCAGGGGATCCCACCTCTCAGACTGCGCTCCTG
This mutation is a 342 bp deletion beginning at Chromosome 12 position 83,315,268 bp and ending after 83,315,609 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49935 | GGA TCC CCT GCC AAA CTA C | Common | A | |||
49936 | AGT CAC GCA AAC GCA CCA | Wild type Forward | A | |||
49938 | GAT GAG CCA GGT GAG TAG CTG | Mutant Forward | A | |||
49939 | Fluorophore-1 | CAC GCC GGG CTT TGC | Quencher-1 | WT Probe | ||
49941 | Fluorophore-2 | CTC AAG TTT TCC TGC TGT GGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49935 | 0.40 uM |
49936 | 0.40 uM |
49938 | 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 |