Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:13954816-13954947 132bp TGCCAATTGTGAGGGTAGAG TTTTACGTGCATCTTGACACA
Mutant= 118 bp
Wild Type = 132 bp
TTTTTTTCTTTTTTTGCTTCAAAAGTAGTTCTCTTAACAATTATTGACTTAGCCTTTCTGATATACCTGAAAATTTCCTCTCAACAACCCATAGAATGCCAATTGTGAGGGTAGAGTTTTACCTAAGACCTCAGCCCCTAggacaggcagatataccttggacaatcacttttctttgaagagaaaaatagaagaaagatttttaattgtgtcaagatgcacgtaaaatttatcatttttgtgttcacctcaactgatggcattaaatgtatttatttttcagtaattttcttttcattgagcaaatgagtagttatatactgttgtcttgagagttgctcagaaccaaatattcgttttaattctcattcatcttttttcctccagacgaagttatacggaaacgtcttctgattgatggagatggtgctggggatgatcggagaattaatctcctcgtgaaaagcttcattaaatggtgcaactctggatcccaagaggaagggtactttattccactgacgcttctgtatcagtatcctggtggaactttattaaggccatgttaaatagttcaatttaccttatctgtccagtgcggccacctctagactgatcagaagtttggtttatttatttgtttgtttttgttttttttttttttgttttttctgttcttctatttggaggtctcctttatgtttgtataATATGCAGATAGGACATTAGTAAAAGAAGAAATGAAGGCTTAATAATTTGATTACTTTTAGCAAAGGAAGGTTTTTTCTTTGTTAAGTTAAATTTAAGTTGCTGCAAAATAATTATATACTAATAATTACATACTGTATTAGTTAGGGTTTTACTGCTGTGCACAGACACCATGACCAAGGCAAGTCTTAT
A 569 bp deletion beginning at Chromosome 14 position 13,954,335 bp and ending after 13,954,903 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44371 | TGC CAA TTG TGA GGG TAG AG | Common | A | |||
44372 | TTT TAC GTG CAT CTT GAC ACA | Wild type Reverse | A | |||
44373 | AAA CCT TCC TTT GCT AAA AGT AAT CA | Mutant Reverse | A | |||
44374 | Fluorophore-1 | ACA GGC AGA TAT ACC TTG GAC AAT | Quencher-1 | WT Probe | ||
44375 | Fluorophore-2 | AGC CCC TAA TAT GCA GAT AGG AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44371 | 0.40 uM |
44372 | 0.40 uM |
44373 | 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 |