Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:129978374+129978501 128bp GCGCAAATCAGTCATGGA CCAGTAACCCTATATTTGAGATTCC
Mut= 130 bp
Wt= 128 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGCTTTCCCTTGCAAATGCTAATACTGTCAGTAAAGAAACAGGGTGCCTACCTTAGGAAAAGAAAGTTTAGAGAGTCTTAGGTGGAAATACAGAGCTTTTAGGGATATTTGTATTAAACCATCAATATAATTCAGACACAGTACAGAAATCTAATGTTTGGCCTTACCCTtgcgcaaatcagtcatggaaaaaaagatatttaagtgttttgcaaacatagttgattgtggaattggataatattctcaaataattttacatttaaaaaattagggaatctcaaatatagggttactggttttccttattttacagatggggtcgaggatttggtcttttgggttccatttttggaaaagatggtgtattaaaccagccaaacagtgtctttggacttatattttatatactacagttattacttggtaagtattgatacagagatacagaaataagcttgttatacccattcagtgcttgtgtcttcaccctagTGTGTCTTCATAGTAAACGTACACGCTAGTAATAGAGAACCACGAGAGTGAAGTGTGAGTCACTGCTGCTTATCATGTGTTGACCTTGGTATCAGTGGACGTTTAGTCATGGCTAGTAGTGTGTCATGATGTCCAGAAACCCAGCCACATTCATGGAAATT
This mutation is a 325 bp deletion beginning at Chromosome 5 position 129,978,373 bp and ending after 129,978,697 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44580 | GCG CAA ATC AGT CAT GGA | Wild type Forward | A | |||
44581 | CCA GTA ACC CTA TAT TTG AGA TTC C | Wild type Reverse | A | |||
44582 | GTG GAA ATA CAG AGC TTT TAG GG | Mutant Forward | A | |||
44583 | GGT TCT CTA TTA CTA GCG TGT ACG TTT | Mutant Reverse | A | |||
44585 | Fluorophore-1 | AAA CAT AGT TGA TTG TGG AAT TGG A | Quencher-1 | WT Probe | ||
44587 | Fluorophore-2 | CTA ATG TTT GGC CTT ACC CTT GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44580 | 0.40 uM |
44581 | 0.40 uM |
44582 | 0.40 uM |
44583 | 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 |