Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:106709641+106709784 144bp TTTTATGAGGCCACAGTGCT GGGGCCATTATATCTCACAGG
Mut = 143 bp
Wt = 144 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ACAAAACTATATTTCAAGTGCTTCTCGTTGTTTTGCATGCAATCTTATATCATTTTTTTCTTCTCTACTTTACTCCAGGAATCCTGCCCAACTTCACATTTTAAAAAATCGAGACCATAtgaaagacctccgcaggtacctgatgttcaaatacatccagtatctagtcctgccttctccgaatgtcatcatcattctgctgggatctcttgccagtctctatgtcatgctgctattgacatttccttccgtttccagaaaatccactgcggtgtttattggtagcatagctcaggcagacatcttagttgtctgcagcttgttttctgctatctcagcatgcgtgataaggagcgagccatcgtcaacttcttttcaactagccttgaggcaaaacttccaaattgcaaatatccatgccagttcccttttactcagctgtgttacccttgaggcttttctgattacttttcttccagtagagacacgccacataaggaatgttagatgtgccagagtggcttctaaaatcatctgggctgttgtaattaccgagtgtttcctttatcagcttgaatatgtcaaaggccttaacatctcctatcttggcattcataggcaaattcagctattgatgaatttcttttatgaggccacagtgctgctgaaattacttatttatcccattggagttcttctaagaatcttcaacgtatatcttttttataaaatgtatttccgagaccatTATAGTTAGTTTCTCAGGCCTGTGAGATATAATGGCCCCGTTGTTTTGTCAAACAGTCATATTTATGGGGCTATGAAGACATTTTATAGCTGTGGTT
This mutation is a 629 bp deletion beginning at Chromosome X position 106,709,117 bp and ending after 106,709,745 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51149 | GGG GCC ATT ATA TCT CAC AGG | Common | A | |||
51150 | TTT TAT GAG GCC ACA GTG CT | Wild type Forward | A | |||
51151 | AAG TGC TTC TCG TTG TTT TGC | Mutant Forward | A | |||
51152 | Fluorophore-1 | CCC ATT GGA GTT CTT CTA AGA ATC TTC | Quencher-1 | WT Probe | ||
51153 | Fluorophore-2 | CTT TAC TCC AGG AAT CCT GCC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51149 | 0.40 uM |
51150 | 0.40 uM |
51151 | 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 |