Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 97 bp
Wild Type = 114 bp
>chrX:107103373+107103486 114bp CTATCTCGTCATGGAAGCTC GTCAGGTAAAGAGAATCCTAACT
Wt Sequence (deletions in lower case; insertion with carrots^^):
TATTCTATATCTCAGTGTAAAAGAGAGCCCTAGATATTCACTAAAGTATTATTTAACCATTGTGCCTTTAAACTTTGTGTGTTTTTTTTTACAGGAGCCATAATTCCACAGTTTTCCTTGccaaacatactgaaatattcaA^a^GatgggcagcaacagtaccagcagtgctgagagcaattgcaatgccacttatttgccatttcagtactctctgtatgcaactacctatatcttcatattcatcccgggtctcctggctaacagtgcagccctgtgggttctgtgccgcttcatcagcaagaagaacaaggccatcattttcatgatcaacctctcagtggcagaccttgctcatatcctgtccttacctcttcggatttactattatatcaatcgtcactggccattccagagggccctttgcctgctgtgtttctatctgaaatatctcaacatgtatgccagcatttttttcttgacatgcattagccttcagaggtgcctctttctcctcaaaccattcagagccagaaactggaagcgtaggtacgatgtgggcatcagtgctgtcatctggattgttgtagggactgcctgtttgccatttcccatcctgagaaatgctggcttagccaatagcacagattcttgctttgctgatcttgggtacaaacagatggatgcagtggttctggtcaccatggttgtaattgctgaacttgctggatttgtgattccagtgatcaccattgcatgttgtacctggaaaacaactgtatccttgaaacacccaccaattgcttttcaagggattagtgagaggaaaaaagcattgcggatggttttcatgtgtgctgcagtcttcgttatctgcttcactccttatcacattaacttcattttttacaccatggtaaaggaaagcatcattaccagttgtcccactgtaaaaagtaccctgtatttccatcctttttccctatgccttgcaagtctctgttgtcttttggacccaattctgtactatttcatggcttcagagtttcgtgaccaactatctcgtcatggaagctctgttactcgttctcgtctcatgagcagggagagTGGATCATCAATGGTTAACTAAAATGAGAAAATTCTTCAGTTAGGATTCTCTTTACCTGACATTCTGTGGCATTGCCCAAAGGCAGGAATTTCCAGGCAAATAATTTCTTTAACTGAACCTTGGAA
This mutation is a 988 bp internal deletion of region ChrX:107,102,438-107,103,425(GRCm38/mm10) and removes most of exon ENSMUSE00000383594 and has a 1 bp insertion at ChrX:107,102,459.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
71609 | CTA TCT CGT CAT GGA AGC TC | Wild type Forward | A | |||
71610 | GTC AGG TAA AGA GAA TCC TAA CT | Common | A | |||
71611 | TAC AGG AGC CAT AAT TCC AC | Mutant Forward | A | |||
71612 | Fluorophore-1 | TGT TAC TCG TTC TCG TCT CA | Quencher-1 | WT Probe | ||
71613 | Fluorophore-2 | TTC CTT GCA AGG TGG ATC ATC AAT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
71609 | 0.40 uM |
71610 | 0.40 uM |
71611 | 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 |