Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:90222311-90222405 95bp CTGAAGCTAACCATGCAGTAG TTCTGGTTGCAAGTTTTCCA
Mut= 90 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGGGCTGGTGGCACACAGTGTCCTGTGTTCTTCCATGTCAATTAGTGATTAAAAAATGCCCCATAGGCTAATCTGATGGGTGAAATGTCTTAGTTGAAGGCCCTTTCTTCTGGATGTGCCAAGTTGACAGCTGAAGCTAACCATGCAGTAGTTATCAGTAAAGATCCTTTAtgaactcatcaaacaacttttttctttagttaggtggaaaacttgcaaccagaaataaaagacatcgccaagcgccttaaccaagaagttgcagtctgtgcccgtcagtttggatggtccaagagagtggctaggtatgcagctttgggtctgcggtcacgaaaatgtgtgtgtggtgtgtgcgtgtatacacggttaggtattcttttgattggcgtattcatattaatcactatgctgcATGGTGGTTTTATGTATTGGAATGGAATCAAAGAAAGGAAGAAACAGGCGTTATTCCAACTCCTGTGAAGCTGAATCGTCCTCCTATCTCAGCCTCTCTCTCAGCTCTGAGATTACAGGTGCAGACCATTGTATCTGGCTCTGTATGTCAGAATATGACAGGGTTTCATGTGCACCAGGGATGCCCAGTGG
This mutation is a 241 bp deletion beginning at Chromosome 5 position 90,222,124 bp and ending after 90,222,364 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45575 | CTG AAG CTA ACC ATG CAG TAG | Common | A | |||
45576 | TTC TGG TTG CAA GTT TTC CA | Wild type Reverse | A | |||
45577 | GCC TGT TTC TTC CTT TCT TTG A | Mutant Reverse | A | |||
45578 | Fluorophore-1 | CAT CAA ACA ACT TTT TTC TTT AGT TAG G | Quencher-1 | WT Probe | ||
45579 | Fluorophore-2 | AAG ATC CTT TAA TGG TGG TTT TAT GTA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45575 | 0.40 uM |
45576 | 0.40 uM |
45577 | 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 |