Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 95 bp
Wild Type = 98 bp
>chr2:120666704+120666801 98bp GGACTAGCCCATCAGGTTCC TGAGCCAACAAGGGTTAGGA
Wt Sequence (deleted region in lower case)
GAGGGTGTTCGTTCATGTGTCCAGTGTCTAGGGGTAGCAGTAGGGATCTAGAAGGGACCACATTAGTAGTGTGAACTCATCTAAGCACGATGAAGTTCACTGCAGGAAAGCATATGCCTAGGTTTCCTGCTTCTCTTATTTGTCTTATGGTGATAATCTGCCCCTCGTCTGTATgtccgtcccttttgttaGTCTCTCCTGAAAGTGCCAGCAAGTAGTCTTCTATACACTGCTGTATAGGCTAGCTCGCACACACAGCCACCCTGTGTGCTCAGGGACTCCAGGTGctttgtggtaagatgagggcatgtgggcagtgtgggtcaacagtaatgctttccttttcttgtcaccaggcctctgttgggctgactccgaggatatgtgaggtatagattctctttgggtcacgtcctccccccgtgggcttcctatgtcagcttccttcagtaaagcctaacgctctggtctgtgcagggtctcttcatcagggaggatgactgtgcctctcagccttgttcccgtagcataaaagtaaggtaagagctcaagccccagaccccagatgtcccatttccatccccagctcctttcacgtcagggtcagcagaggggtggagaaaatccgcagaggacacatcatgcagtataggaaatgggacacatactaagcctttgggttgccccggactagcccatcaggttcctcctgtgcctctgttcccctcacttcctgttgtCTCACTCTTGCTTTTGGGATTCCCCTCCTAACCCTTGTTGGCTCATTCTGGAGATTTCAGACCGACTTTCCAGACTAGTGCTAGGTACAAGGTTTTGTTTTGTTTTGTTTTGTCAGACAGCATCATGCTATGTAACCTAGGCTGGCCTCAGACCCTTTATCCTGCTGTTTTACCCTCCCAAGTGTTGTAGTTATAAGCATGATCCTCTATGGCCTAGCAAAACAAAGTTTTACCAAGCTGCATTGAGAAAAATATTGATATATGATTACTAGGTTTT
453 bp deletion beginning at Chromosome 2 positive strand position 120,666,304 bp and ending after 120,666,756 bp (GRCm38/mm10). In addition there is a 17 bp deletion (GTCCGTCCCTTTTGTTA) 96 bp before the 453 bp deletion
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38198 | TAG GCT AGC TCG CAC ACA CA | Mutant Forward | A | |||
38199 | TGA GCC AAC AAG GGT TAG GA | Common | A | |||
38200 | GGA CTA GCC CAT CAG GTT CC | Wild type Forward | A | |||
38201 | Fluorophore-1 | CCC TGT GTG CTC AGG GAC | Quencher-1 | MUT Probe | ||
38202 | Fluorophore-2 | TGC CTC TGT TCC CCT CAC T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38198 | 0.40 uM |
38199 | 0.40 uM |
38200 | 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 |