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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:117385436-117385539 104bp TGACACTTCCAGCCACTAGC ATATCGATCCCTCGGTCGTC
Mut = 97 bp
Wt = 104 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AATGGCTGGCTGACACTTCCAGCCACTAGCTTGTAGCTGGCAcagggtgtgactgtcacagcatcctctcttcctcctgtagctgaatgcttctgacgaccgagggatcgatattgttcgggggccaatcctcagctttgccagcacaaggacaatcttcaagtaagaattacctgtaggatttctctccctacaggtcttagtgcgggttaGGAGGGACACAGCCTGGCTGTTCGGTCCCCCTTGGCTGGTATAAAGGGAAGAAGGGAACACCTGGACAGAAGCAG
This mutation is a 170 bp deletion beginning at Chromosome 5 position 117,385,338 bp and ending after 117,385,507 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51198 | TGA CAC TTC CAG CCA CTA GC | Common | A | |||
51199 | ATA TCG ATC CCT CGG TCG TC | Wild type Reverse | A | |||
51200 | CCA GGT GTT CCC TTC TTC C | Mutant Reverse | A | |||
51201 | Fluorophore-1 | CTG TCA CAG CAT CCT CTC TTC CT | Quencher-1 | WT Probe | ||
51202 | Fluorophore-2 | AGG AGG GAC ACA GCC TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51198 | 0.40 uM |
51199 | 0.40 uM |
51200 | 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 |