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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:38999897+38999987 91bp AGGCCTTGTGGTTGGAACT AAGACTTCACTGGGCCTGAG
Mutant= 93 bp
Wild Type = 91 bp
Wt Sequence: aggccttgtggttggaactacagataacaaatctggacCtggagggcaggacagtgccttgtgttctctgcctcagGCCCAGTGAAGTCTT
Mut Sequence: aggccttgtggttggaactacagataacaaatctggacCCtcttagtgcctcccctactctctccccacacccaatcagatgcaggttccagc
A 381 bp deletion beginning at Chromosome 2 position 38,999,936 bp and ending after 39,000,316 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65551 | AGG CCT TGT GGT TGG AAC T | Common | A | |||
65552 | AAG ACT TCA CTG GGC CTG AG | Wild type Reverse | A | |||
65553 | GCT GGA ACC TGC ATC TGA TT | Mutant Reverse | A | |||
65554 | Fluorophore-1 | TCT GGA CCC TCT TAG TGC CTC | Quencher-1 | MUT Probe | ||
65555 | Fluorophore-2 | ACA GTG CCT TGT GTT CTC TGC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65551 | 0.40 uM |
65552 | 0.40 uM |
65553 | 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 |