Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:56913179+56913269 91bp CAAATGTTTCACAGCAAATAGACC GGCAACAGTGTGGATGAATG
Mutant= 111 bp
Wild Type = 91 bp
X-Linked
Wt Sequence (deletions in lower case; insertions with carrots ^g^ (g insertion):
CAAATGTTTCACAGCAAATAGACCTAACAACTTCTTCCCAA^g^actactggacttaacccacacaagacttcacattcatccacactgttgccagaaggcatggctgattctacaatcaacagtacagccatatcatatgctaatacagtaccctcacctctagcaacagtaagtgcagccaaagatttgaagacatctacaaccgagacagctacatttttaacagataccttgtttacttcaacagccacccctctgcctacccagactgtaacagaacactcatatctaggaaaaacaaggacatcaaaaagggtggaggccatggctactgagatctttcattcagctacagctactgatttaattgacacatctgtgtttactaagaattatactgtatctgaaacttcaacaactaagtccaagtcagctgttgggaagacaactttattcttgaatgaatcaacatccatagctccaacaccttgtcccaagcacaaatccacagatgttgctatactccatacctctaaatctgggcaagaatttcttgtatcttcagctgccagaactgtgtcttggtccacattagaagagacttcacctattactactgatgttgggattgtatcaaccttcccaccagagtctttgcttacctccacagcttctcctgtgagttctacatttcctgaaattcagttagcatctaccttgtcaacaactgattcagaaatggcatcaacagtccattcagtgttgcctatgcagaccatacctaccccaagaacagtgaaaccagagtcaggctctacaaatttccaagatgtcttttcacccagcatggaggatgctctatctgtctccatgcctaaagaaaccactttcatggccttttcttccattacctcctctccaatcaccagaactcaagatgaacagatagctattgatgctgagagtacacatctaaccataatccccggaaccaaatttgtccctacattggctgaagcttcactcttccctaccattgaagggcaagcatacactcaggatacacccacaacggatgagcccatgcttactttgacttctactaaatcaccttctacatataacgcatctgaatcagttctcacatctataacaattaaaagtgactatcagttcttcaccaatgagaccacttggacttctaagtcaggtcagaacctgttgacctctatgaatacaaccactatacccacattcacatctaataaaactctcacattaccattccaaggtaatgccactaatagagatcattcatccatgactactaacgtctcaccaattgaagcatccacggagagcaaagtgactaccagctctgatgctactacagccagttatacaacagctttattcaaaccaacatcccagtggcttagtcatttcacgagtgtttctggaatcacatctatagccagccagcctgaatctaaactcaccaccttattattgaaaagcaactctatgcccacagtagctacaaatgaatttccctcaataccaagtgagccagttgctccatcagtgaatacgtctaccctcactgacattaaaccaaatttttctacagagaagagcatttctgagacaatacaaatagaaacaaatggtgtgagctcatttggtgatactctggcccctctgcccatgtctgcaacaacacagagagtctataccactgtgacaaaagaaaccacttcccgtcatcctaaggtaaagtcaactatatcaacagtagctgaggcttctccattttcaacaatgctggaagtgacagatgaatcagaacaaatggtgacagcttctgtcactatttccccctttacagatatagaaaaattgactactgcattgagtaaagaaactgcaactgcagaggtgggagtgagttggctttcaaccaaattaaaggaaagcatgcctgagagttcacacaatggaaccacagaaagctttaactcgacccatacctacacagtagattggacttcagaaaaatccaagggtaactcagcttcatcacccaattctgcaagcacccaggcactacctgaacttccaagttcttcaacaatgaagacgatgggagtcactttctccactaattcttcacagaggacagctgcatctttgtctgctggtatcttgtctccacagactgcctctactcatgccttagtaactccccaactgcttacaca
This mutation deletes 5813 bp of ENSMUSE00000386381 (exon 3) and is predicted to cause a change of amino acid sequence after residue 254 and early truncation 9 amino acids later. There is a 1 bp insertion (G) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49531 | GAG AAC ATG GAG AAC ACA TGG A | Mutant Reverse | A | |||
49532 | CAA ATG TTT CAC AGC AAA TAG ACC | Common | A | |||
49533 | GGC AAC AGT GTG GAT GAA TG | Wild type Reverse | A | |||
49534 | Fluorophore-1 | CTA CTG GAC TTA ACC CAC ACA AGA C | Quencher-1 | WT Probe | ||
49535 | Fluorophore-2 | CAA CTT CTT CCC AAG TAC AGG TAT TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49531 | 0.40 uM |
49532 | 0.40 uM |
49533 | 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 |