Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:30608167+30608267 101bp CTGCCTCCTCTGCTTCAGAC AACGTACATCCTCTGTGCTTCA
Mut = 97 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CATGACAGCAGCTGAGAACGCATCTCCCTGTCCCAGGTGGGGCCTGCCTCCTCTGCTTCAGACTTGCTTGGAGTTCGGAGCTCAGctgagccataacggaggctcacaaggaagagatcctgtgaagcacagaggatgtacgttaaggagttggggtgtggccagatatctgcctggggaattctgtactggaaggctctcccagctactcaggcaatcctgtgcctgtctccatggctttcaggagcatctcttgggtgacctggctcaccacatagtttcactgagagccatcatgagttggtacagccagTGGCCTGCCTTTCAAGTTCAAGGTCAGCCTTAGCGATGGAGAGAGAGAGTTCAAGACCAGCTGGACTACAGGGGAACTGTCTCAAAACAAACAAAAATGAGCTAAGAGGTTAGGAAGATGGCTCAGCAGATAAAAGCATTTG
This mutation is a 228 bp deletion beginning at Chromosome 2 position 30,608,209 bp and ending after 30,608,436 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51310 | CTG CCT CCT CTG CTT CAG AC | Common | A | |||
51311 | AAC GTA CAT CCT CTG TGC TTC A | Wild type Reverse | A | |||
51312 | CTT GAA CTC TCT CTC TCC ATC G | Mutant Reverse | A | |||
51313 | Fluorophore-1 | CCA TAA CGG AGG CTC ACA AG | Quencher-1 | WT Probe | ||
51314 | Fluorophore-2 | CAG TGG CCT GCC TTT CAA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51310 | 0.40 uM |
51311 | 0.40 uM |
51312 | 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 |