Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:140883371+140883455 85bp GGCGCTTGCTTTAACTCCT AAGGATGCCTTCAGCCTTG
Mut= 89 bp
Wt= 85 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TAGGTTGTGGCATTGGAATTCCAGTTGACACTCAGAGTGGGAGTTGACGCTGTCTCAAAAAGTGTTCTCCAGTGTTACCTAAGAAAACTGAGTCTTGAAAGGATAAGTGACTAGGGGGTGCAAGCAAAGTCAGTCCAGAAATAGGCTTGAATTGGCTGGCGCTTGCTTTAACTCCTGGCTgtgcagtgggacctgggaaatacttggtgtttgatgggaccgtcaaggctgaaggcatccttttcttcacattaggatgtgggactgagggctttttgttttgtaggttgtggcatcagctgacacttgaggtgcttgattttgtgcaggacccatgctttgcccaaggagatggtctcattaaggtaatgacttaccagacagctcaGACGATGTTGAATATAATTACAAACTATTATTATTCATTTTTATTTACTTTTAACGTGTGTGCGCCCAAGGATGCCAGAAGAGATCTCCTGGAACTGGAGTTGCAGGCAGTTGTAAGCCAACTGGTGTAGGTGCTAGGAACCCAGTTTG
This mutation is a 208 bp deletion beginning at Chromosome 7 position 140,883,394 bp and ending after 140,883,601 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41206 | GGC GCT TGC TTT AAC TCC T | Common | A | |||
41207 | AAG GAT GCC TTC AGC CTT G | Wild type Reverse | A | |||
41208 | GGC GCA CAC ACG TTA AAA G | Mutant Reverse | A | |||
41209 | Fluorophore-1 | CAG TGG GAC CTG GGA AAT ACT | Quencher-1 | WT Probe | ||
41210 | Fluorophore-2 | TGG CTG ACG ATG TTG AAT ATA ATT A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41206 | 0.40 uM |
41207 | 0.40 uM |
41208 | 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 |