Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:56723728+56723860 133bp GGTGAGACATGCACAAGAGA ATTGAAGGCACACCCATCAG
Mut= 118 bp
Wt= 133 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (T insertion):
TTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCATAATGAGATCTCATGCCCTTTTCTGGAGTGTCTGAAGACAGCTACAGTGTACTTACATATGATAAATAAAAATAAACCTAAAAAAAAAGAAAGAAAAAATTGTGAGTGTTCATGTAGAAAGGAGGTTAACTGTTTTGAAATCAATTAGCAAA^ctcatctaatttttaagtacaagttccaattgtagtaattttaccagcaggaaacaagaattctaaaaactgccaacataaggtaggcagtctacaatttatggatgaattgtgtttccagagttgctatttaaagcctggaccacattttcccaaagaaactgtgttacagacaccgtgaacccaattcttcctgacacggtcttaatttcacttacagaattcttgagtatgtttagcacctaaactctatttaaacatactcaactctatttaaactttgagttacaactagaccctttaaatcagctgggaatagtctataaaccttcccatctttctccacttctgcttttgagtttgttgataatcttcaatagcatcattcagggtttaagcacatgacagttcatcagtgtaagaccctttctttgatcactgtttggctaacattctttttcattttaggttgaattctcttacccaccactaattccaggagatggacatgatagccacactttacctgaagaatggaagtacttgcctttccttgccttaccagatggtgcacataactatcaggaaggtatgtcattaaatcgtagaccaattactaagtgcaattttggaaattttctcatagcggtcgtgaatactttggataaatgaaagcctttggacctgttgtgattacttataaaatgtgtatgtgttggaggtgagacatgcacaagagaagcttcagagagtactgggcctatttccttggttagtaaccctcgg^CTCCCTCCTGCCCAGAGCTCTTAGGTATAGCCAGAGCAGGGCTGCTGCTGATGGGTGTGCCTTCAATTATATTATGCTTGAGACTGAACTCAGTGGCAGAAATGAAGATTTGCCTGCATTAGCTGTACATGGTCTGGAAAGCTCTGGTTTCAGACTTACTCAACCATCATCCTCTAATGCCCACCTGGATACAGTGG
This mutation is a 786 bp deletion beginning at Chromosome 6 position 56,723,008 bp and ending after 56,723,793 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47753 | GGT GAG ACA TGC ACA AGA GA | Wild type Forward | A | |||
47754 | ATT GAA GGC ACA CCC ATC AG | Common | A | |||
47755 | TGA GTG TTC ATG TAG AAA GGA GGT | Mutant Forward | A | |||
47756 | Fluorophore-1 | ACT GGG CCT ATT TCC TTG GTT A | Quencher-1 | WT Probe | ||
47757 | Fluorophore-2 | CAA ATC TCC CTC CTG CCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47753 | 0.40 uM |
47754 | 0.40 uM |
47755 | 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 |