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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:93445233+93445363 131bp AGGAGCGAGAGAGACGTCAG TCGCTGAAACTGCTCTTCC
Mut = 119 bp
Wt = 131 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
ggctaaaccatctctccagctcctctgtgcattttctaatcgctccaaatagtcccctccCAGActcatatatagtctggaaattcatgtattcccaatatcttatggtagttttactattagctatgtccaaataagaaaggaaga
This mutation is a 5805 bp deletion beginning at Chromosome 3 position 93,443,307 bp and ending after 93,449,111 bp (GRCm38/mm10). There is a 2 bp insertion (AG) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52168 | AGG AGC GAG AGA GAC GTC AG | Wild type Forward | A | |||
52170 | TCG CTG AAA CTG CTC TTC C | Wild type Reverse | A | |||
52171 | TGC ATT TTC TAA TCG CTC CA | Mutant Forward | A | |||
52172 | CTT CCT TTC TTA TTT GGA CAT AGC | Mutant Reverse | A | |||
52173 | Fluorophore-1 | AGG AAG AGA AGA GAG AGC TGG AGC | Quencher-1 | WT Probe | ||
52174 | Fluorophore-2 | CCC TCC CAG ACT CAT ATA TAG TCT GGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52168 | 0.40 uM |
52170 | 0.40 uM |
52171 | 0.40 uM |
52172 | 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 |