Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:31626957-31627068 112bp CCACTACTATCCATTTACAAACGT CCAGCCTTTTAAATAATGGACTTTAG
Mut= 113 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
AGGCTGGATTATATCTGTTGCTGACAGCAAGTTGGATTTTAGTCATGCATAGAAATGGAAAGAGTCAGAGATAATATTTCACAGTAGATCAAGACTTGACAGTAAGATGATAAGTGAGCCCCAGACGAATGATTTTAAAATGCTATAGTGCTGATTAATAACTTGCCACTACTATCCATTTACAAACGTCCATCTTTAAGCTCAATACCCTGTCAGGTACtaaagagggacagacagaaagaaaggcatatctaaagtccattatttaaaaggctggtaggttattctacaactccttcttgtttaaagaaaatgctttcttaaaaactaaaagtagcgtcctctgtcttgtaacagatgccgtgagagaagtaagaaagtattcgtccacgaatgttgtagagaagaactcagccatcagaccgagtgcctttgagcacacacagatgaagctcttcaggtctcaaagaaatctgtatatttctggattctcattatttttttggCTGtaagtaaaaaaaaatttgctagcagttgtatcactcatagaactcccctttgttttttccacttacACTAAGATCTTTTCATTGATATCTTCATAAAAGAATGCAAATGATACACTCAAGTAACAAATCTCAGTCACACTAAAGAACGCTTGGAATGGAAGATGTTGCCTTTGGAAGGGAAGAGCCCATGAATTGGTTGTCCAATGCCAAATGTTCAGCCCTGAAAACATACATCCAGGTAGCAGTATATGGACTGAACAGGCTATATTTAGGAATATATATGTATG
This mutation is a 355 bp deletion beginning at Chromosome 12 position 31,626,659 bp and ending after 31,627,013 bp (GRCm38/mm10).In addition, there is a 3 bp endogenous retention (CTG) 286 bp after the start of the deletion and then an additional 66 bp after the 3 bp retention.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46975 | CCA GCC TTT TAA ATA ATG GAC TTT AG | Wild type Reverse | A | |||
46976 | GTT ACT TGA GTG TAT CAT TTG CAT TC | Mutant Reverse | A | |||
46977 | Fluorophore-1 | AGG GAC AGA CAG AAA GAA AGG | Quencher-1 | WT Probe | ||
47858 | CCA CTA CTA TCC ATT TAC AAA CGT | Common | A | |||
47917 | Fluorophore-2 | AGG TAC CTG ACT AAG ATC TTT TCA TTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46975 | 0.40 uM |
46976 | 0.40 uM |
47858 | 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 |