Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:173572614-173572742 129bp CCCCTTTGGCTCAAGATTTTA TCAGAATTCCTACGGGAAACA
Mut= 96 bp
Wt= 129 bp
Fam=Mut
Hex=Wt
The Mut Reverse primer (primer 64361) anneals over the nucleotide sequence containing mouse genomic variations rs223620354.
Wt Sequence (deletions in lower case):
TCTTCTTTTTGGGTCCAGCTTCCATTTGAACTAAGCCAAATTAATTTTCTAAAAAGCCTTAGCAGCATCGAATTTCAGGCTCCTGTGCGACCATCAAGGGAGAGACTTCCCCCACCCCTACCCCCACGCACTCCCGCCCCTTTGGCTCAAGATTTTAAAAGTATCTTTGCGTGATTCAGGGCCAGacgtggtctgaactgatgcagtgatttctctaggtcctaattttgttaaaaggaaaaactgtttcccgtaggaattctgatggatgatgctaccctgaagttctacgatgtcatccccggagcacttatttcattatgtatctggcattacgatggttggacggaactggttttggccgccgtggaaggggaccccagcaaggtggtttcgtgctttttttttcaatattagggggcgaggggaccccagtctttagtctttagtgtgaaacttttgaataaagtcctcagagtagggaaggaagaagagctttctgcatggagagtgtttccagacagtctgggcttgtgagtccctcgGGCTTGCTTATCAACAGTTCATAAACCCACTTACTCTACCCGCAGTGGGGCCCAGGTGCTGCATTGTCTGTCCACATGTTGGCCAAGTTGAAAATCGAATTTAATCCTAGTTAAAGAACATGGGGTGGTAGCCCAGTGGTCAGGGGCTTGTGTACTCAGGTTGGTCTACCTTGGG
This mutation is a 360 bp deletion beginning at Chromosome 2 position 173,572,334 bp and ending after 173,572,693 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64359 | CCC CTT TGG CTC AAG ATT TTA | Common | A | |||
64360 | TCA GAA TTC CTA CGG GAA ACA | Wild type Reverse | A | |||
64361 | CAC TGC GGG TAG AGT AAG TGG | Mutant Reverse | A | |||
64362 | Fluorophore-1 | CGT GGT CTG AAC TGA TGC AGT | Quencher-1 | WT Probe | ||
64363 | Fluorophore-2 | CCA GGG CTT GCT TAT CAA CAG TTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64359 | 0.40 uM |
64360 | 0.40 uM |
64361 | 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 |