Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:59727279+59727362 84bp TAGGATCTTGGCTGCCTGCT AACAGTGTCCTGGCTTGCAC
Mut= 86 bp
Wt= 84 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGCCTTTGGGTCTGTCTCCCAATCTTAGAGCTAGTGCTCAAAGATGTGGCTCTATTTACCTACCTCCTTACGTGTTTTTTTGTTTGTTTGTTTGTTTTTTTTTTTTCTATTTTGAATCTACTATTCCCGAAGCTTTTCCTCACTGATCTGAGACTAGGATCTTGGCTGCCTGCTCTCCCTagcattcctcactgagcactgtcctgacccctcctgcggtgcaagccaggacactgttctccatgtagaaaataaagtccagagggtgtgtcctctacataagtccagcttggcttgccccaccgtggatgggtgggtaggaagtggtgctgtgaaggctgaggactgaaggcaaggggtgggtcctgctggtggctgtgggacctgacacaccaccccttttgagtgacttgttttgtccttgcagcccaccaccctccctcagggcaccatcaacatgaaccagtgcacagatgtggtggatggggaggcccgcacagggcagaagttctccctctgcatcctgacacctgacaaggaacattttatcagggcagaaaccaaggagatcatcagtgggtaagttggccagagccacaggaggcactgtctgtttgctcacccatttctggggactccagggcttctaggtcttccttggaactcttcactgattggtcgtcctcccaccccacacctcatcctcaccctgtgcctccttggcactcagcacacagtgctgctcctgccttcccctcaagtaaaataagccctcttttctaggctgtgtatcaggcagcctgcgtggcggcccctttgagaaacgtcccgtgtaGCACCAAAGCCTCCAGGTGATTCACATGCATGTATTTTCGGTCCTCAGTGATTAACCCCATTCATTCTCTACGGCTGGAAGAGGGCAGGTGTGTCATCAAGGCTCTGTCTGAGGTTGAGGGGTATAGGCTGTGTATAGCACAGGGTAGAGGGAAGTAAGGACATGAA
This mutation is a 655 bp deletion beginning at Chromosome 11 position 59,727,305 bp and ending after 59,727,959 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39075 | TAG GAT CTT GGC TGC CTG CT | Common | A | |||
39076 | AAC AGT GTC CTG GCT TGC AC | Wild type Reverse | A | |||
39077 | TGG GGT TAA TCA CTG AGG AC | Mutant Reverse | A | |||
39078 | Fluorophore-1 | ATT CCT CAC TGA GCA CTG TCC T | Quencher-1 | WT Probe | ||
39079 | Fluorophore-2 | CCT GCA CCA AAG CCT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39075 | 0.40 uM |
39076 | 0.40 uM |
39077 | 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 |