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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:69561020+69561134 115bp CAGGTGAGAAAACTCCCCTTA CTGAAAGTGTGTAGAAACCAAAGC
Mutant= 84 bp
Wild Type = 115 bp
Wt Sequence: caggtgagaaaactccccttatattgtgagaatatacagaatcattattaCagaggccccaggaaggaagagtgtagccttaaatctcaatgctttggtttctacacactttcag
Mut Sequence: caggtgagaaaactccccttatattgtgagaatatacagaatcattattaCTatggggctgaagtacactggcatttgtttgcg
A 377 bp deletion beginning at Chromosome 5 position 69,561,071 bp and ending after 69,561,447 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66159 | Fluorophore-1 | CCC CAG GAA GGA AGA GTG TAG | Quencher-1 | WT Probe | ||
66160 | CAG GTG AGA AAA CTC CCC TTA | Common | A | |||
66161 | CTG AAA GTG TGT AGA AAC CAA AGC | Wild type Reverse | A | |||
66162 | CGC AAA CAA ATG CCA GTG TA | Mutant Reverse | A | |||
66163 | Fluorophore-2 | TAC AGA ATC ATT ATT ACT ATG GGG CTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66160 | 0.40 uM |
66161 | 0.40 uM |
66162 | 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 |