Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:34426472+34426583 112bp CGATCCATCAGTACCTTTCTTG GCTGCATATTATCTCCCTACCA
Mut= 112 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATCTCTGCTGGGATTGGGGCTTGTCACTTTCTAATTCAGTCATTGGATTAGCACCTAGGGTCAATTCTCAGGCACCTTTTACTCTTATTTGTGAGTTATAATACCTTCCTCAGAGATGGACTAAGATCTTTTTTCATGGACTGTAGGTATGGAGTACAGGTTGCATAAACAGTGCAGTCCTAGGAAGCATTTGGGGTCATGTCAGTGTCTATGAGTTACCTGGCATCAAATGCTCTAGGTGCACAAATCCcataggttgtcacaagaaagacagtggattgtcagtgacactgacaggaaaactgcctgttaactgacatgtgtggacaaatcatggctaagtggtgattctttctgtgtctctacaggctctgtgagcgggacccctatcagctctaccagcggttggagcaacaagcccgggaatatgtgctggagatgaaggtccgcctgcttcggcagctctctgctgcagccaaggccaaggcaccgtctggcttgcagggcccacctcaggcacaccactttgtctccctactcctggaggagtatgctgccctttgccaggcagcacgatccatcagtacctttcttggcactctggtgagagaccatatccacttctttcctggcTGCTGAGGTGAACATAAGAACAAAAGCATTTGGTAGGGAGATAATATGCAGCCAGGGGACTGATCATATGGTTAAGCCCTCCTTGTATGATCACAGAGTCCTTTGCACACAAGGTGTGGTAGGATTGTTAGTTTAATGGAATTTGCTAGCAGAGAGAAGCATTTAGAGAATTAATGTCCAGGAGCCATAGGATGAAAATCTGTTTGGGAAAACCTGAGCACTAGGAAGTTAGACACTAGGATATGGAGGTGAGAACAGTGATACA
This mutation is a 383 bp deletion beginning at Chromosome 5 position 34,426,149 bp and ending after 34,426,531 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48089 | CGA TCC ATC AGT ACC TTT CTT G | Wild type Forward | A | |||
48090 | GCT GCA TAT TAT CTC CCT ACC A | Common | A | |||
48091 | TTG GGG TCA TGT CAG TGT CT | Mutant Forward | A | |||
48092 | Fluorophore-1 | ACT CTG GTG AGA GAC CAT ATC CAC | Quencher-1 | WT Probe | ||
48093 | Fluorophore-2 | CTA GGT GCA CAA ATC CTG CTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48089 | 0.40 uM |
48090 | 0.40 uM |
48091 | 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 |