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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:67228511+67228621 111bp AAAGCTGAGAAAGGGACAGC TCCCTGGCCACATAAGGA
Mut = 116 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
ggaagaagggtggggaatcttttttgcacacagtaagcacttagggaaagtatgatcttattctgtgtcagtATtgccgcaggctcttgctgtgcacatccttatgtggccagggaaggacagattaagtggtaaacagtgcattct
This mutation is a 5123 bp deletion beginning at Chromosome 7 position 67,223,456 bp and ending after 67,228,578 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51005 | TCC CTG GCC ACA TAA GGA | Common | A | |||
51006 | AAA GCT GAG AAA GGG ACA GC | Wild type Forward | A | |||
51007 | GGA AGA AGG GTG GGG AAT CT | Mutant Forward | A | |||
51008 | Fluorophore-1 | AGC TGA CAG GAG CCT CGA C | Quencher-1 | WT Probe | ||
52145 | Fluorophore-2 | TGC ACA CAG TAA GCA CTT AGG GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51005 | 0.40 uM |
51006 | 0.40 uM |
51007 | 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 |