Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:115575875+115575964 90bp GTGTTCTTAACCCCTCTTAGCC AGGCCTCACTGGCTAAATCC
Mut= 90 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
The mutant probe (primer 55233) anneals over the nucleotide sequence containing mouse genomic variations rs49091205.
Wt Sequence (deletion in lower case):
GTAGATCTGCCGGCCACTGCTGCGATTAAAGGTGTGCCTCACTACATGTGGCCTTCCTGCTGACTTGTAGACCACTTATTAGACTGTATGTGAGCAAGTACCTTCCGAGCATCACCAACAGCACAGTTTAAAGCCAGCGCCCAGGCCGGAGAGCAGAGCAGGCAGTCGTAGCCCTgctgagccctgctctgtagggagagggagatgctggggtcttgggcttcttctatccatcgttggtttagcaagtgtctcctgcactcggggcgtttctgttccatgtgcctgttttctccttgagattttacaaggtgcggggcttactagggcgtctggtaccaggatcccagctgacaggactgatgtaagcggacactcaccgctgtgcttgttgttgagactctgtgaactctgctttcagatctgccagagggagccgtgaaagggctctgcaagttgttctgcttgacactgcatcgatataggtgagcccccagagaccagggcttagaccgtgccatacagcgacactgtgtcagagagacagcatggggcagtggggagagcccctagttgttagagccagactcttaggttcatgctcttaactactggatcctgggcaagttacttagcctccctatgcctcagttttctcatctgcaaaatggggatgatagcattttgtacctcatgggaagttgttagtgtgaaggcctgggacactgcctgccttattataatatctcagcgcatgtatgcaatcattagctgcacttataaagctataaaggagtcatggggcatatacaccatgagcatagattctgcccttcttgtcatcctgtgctctttctggggtgaggtgggtttatcaggttcatctctgtcccctaggggcccctgaaatgtaggaccatgagtgtccccccgaagagcagtgcttgacttcagcagccctgtctctgctgaggtcacatgtcactaccccttccttccctctttagagacgcagcctctcgcagagccctgcaggcagccatccagcagctggccgaggcccagccagaggccaccgccaagaacctcctgcactctctccagtcttcaggggttggctccaaagcatgtgttcccaggtacgggcctggggctgcgggcctccggggggtggtgccaacttctcaggtaggctggtgtagagccactggtgggagcacatctggccctcttacctttgtactctgaatggcactagagagcgcctctgagtggggagaggaaccaagttagtagagaaaagatccagtgggcccgaggcactggatgggcaaagctttatatacttgttcatgtacattttatgtgaatgagtctcttactgcacgtatgtctgtatactatgtgcatgcctagaacctgaggataccaggggagggggtcaggtccccgggacagtgggagctgccatgtggttgctgtgagaatcgaacccaggtcctttggaagagcaacctgtgttcttgttttttggttttttgtttgtttgttttgttttttgtttttgtttttttcgagacagggtttctctgtatagccctggctgtcctggagctcactttgtagaccaggctggcctcgaactcagaaatccgcctgcctctgcctcccaagtgctgggattaaaggcgtgcaccaccatgcccggctgcaacctgtgttcttaacccctcttagccgtctctccaacctcatcttgtttttactgtcccaaacatcaagggatGGGATTTAGCCAGTGAGGCCTGGACGCTTGTCCTGTTTATCACTGATGGTGTTGGGGGAGGGTTCATACACACACACCATGCTGTGCCACAGGGTGAACATTGTTGTCCCCCCCCAATCCCCCGTGGAGGCTGGAACCCCGCACTAAGCTCTTCTCCTCCCGCTTCTCCTTTGTAGCAAGA
This mutation is a 1611 bp deletion beginning at Chromosome 5 position 115574333 bp and ending after 115575943 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
55229 | GTG TTC TTA ACC CCT CTT AGC C | Wild type Forward | A | |||
55230 | AGG CCT CAC TGG CTA AAT CC | Common | A | |||
55231 | GAG CAT CAC CAA CAG CAC AG | Mutant Forward | A | |||
55232 | Fluorophore-1 | TGT CCC AAA CAT CAA GGG AT | Quencher-1 | WT Probe | ||
55233 | Fluorophore-2 | CCG GAG AGC AGA GCA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
55229 | 0.40 uM |
55230 | 0.40 uM |
55231 | 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 |