Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:25178073-25178163 91bp CTGTGGGTCTCTAGGGTACG GGTGTGGGAGAAGCCAAAG
Mut = 88 bp
Wt = 91 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AACACCAAGCGAACAGAGATCATAGCCAAACGTTGCCAGCCGCTGGCAGTTCATAGAGGTGTTTAGAGGCTCTGAGACTTTTTTAAAGAACTGTGTAAATAAACGTGTGAGGTTCAGAGTTCTGGTGTCTGGCAGTCCACAGAACCAAAGAGGGTGGTCTTGCCAGAGATTGAGCCGGGCTGTGGGTCTCTAGGGTACGCTTTatctccacggctctcccatgcaagcgtaggatgatgtggtactgtgcgctttggcttctcccacaccctgtgctgtgttctgcctcatccccagagagttgtcatcaggattccaaactgtcttgtctgttcctcttgtttctaggagcggctgcaaagtgaagaaatacgaagctcagcctctcgatttggacgcgtgttcccaggtaagtcagtgacgctggccagagtgggttttgtcccccgtggatgtctGGGTACTCGGCCTTAAGGAACCCAGTTATTAGATTCCTAGTGTCCTAAATGTGCCCAAGAAGACCCTGAAAGAAAAGAAAATCTGGAGGCTGGAGAGATGGCTTGCTGCCCAGGAGTACAGGCGTCTCTTCTGAAGGACACAAGTTCAATTCCCAGCACCCACGTGGCAGCTCACAACTGTCTATAACTCTAGTTCCAGGGATCAACACCCTCACACAGACATACATGCATGCAAAACTCCAGTGCACATAAAATAAAAATAAATCATTAAAGT
This mutation is a 255 bp deletion beginning at Chromosome 8 position 25,177,885 bp and ending after 25,178,139 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48900 | CTG TGG GTC TCT AGG GTA CG | Common | A | |||
48901 | GGT GTG GGA GAA GCC AAA G | Wild type Reverse | A | |||
48902 | TTC TTG GGC ACA TTT AGG ACA | Mutant Reverse | A | |||
48903 | Fluorophore-1 | CGG CTC TCC CAT GCA A | Quencher-1 | WT Probe | ||
48904 | Fluorophore-2 | TTT AGG GTA CTC GGC CTT AAG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48900 | 0.40 uM |
48901 | 0.40 uM |
48902 | 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 |