Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:67263908+67264042 135bp ATGGGATTAAGACTAACACAAAGC CCATCTCAACCCAAGACATCTAGT
Mut= 133 bp
Wt= 135 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (A insertion):
TTCTATGCCAACTGTTGCAACGATTTTACACTTCTCTTTTTAACGGATTATTTAATCCAGACCTAATATCTGATGCTTTATTCAGTTCTGAGTCCTGACTATGTGGGTTTGAGATTTTTCTAAGAATAGACATTATAATTCTCTTGAGAGCCATTAGTCATTATTTAAAGTTGTTTTAAAGTAGATGGAGCCATTATTATAAATGTTGAAATAGCACAGAAAGTTTTACTGTCTCGATTTTTTTCTGAAAGTGTTTCTCATGCTTTCCATA^gcatatgtgtgagcaagtggttaagtaacgtgagtttccacttactgatctgcaaaagcaaatattacaagatggtgttacaatattcttcataggattatagtgcataatttgtccttaacatagtttttgggcttagggtaccataatttttttttttttaatctagcaattcatgatgaccaataaattggacacagcaatgtggctttctcgcctgttcacagtttattgctccgctctgttcgttctgcctcttcttgggtatgtattatcttgtttctgactcagaggtgttcggtcagtatctatgactgctttttagctttagaattgttcaattgtaaatgggattaagactaacacaaagcccacttaacagcaacagtaatctggtaataccaattaggatgttttatttctagtttgtgtgactgctcgttgaaaagctga^GGAAAACTAGATGTCTTGGGTTGAGATGGATTCTGCAGGTCTTTTAAGTTGACAAAAGTATGACTGCTTTATACTAATCAGTTGGATGTGTATGTGAAGAGATGTTTCCCAGAGTGTGGAGTACATTTGTGTAATTTGTTTTCTATTGACAATTAAATGATTTAGGTGTTGTGCAATTATGATGCAATTCACAAAGGAGGAAGAGACGTGGTAGCTGTTGAAGTCTCAGAATAACTTTTCCTCTGGTAATTTGCAGGTTGCATGAAGCAGCGAGCTTTTACCAGCGTG
This mutation is a 453 bp deletion beginning at Chromosome 5 position 67,263,561 bp and ending after 67,264,013 bp (GRCm38/mm10). In addition, there is a single bp (A) insert at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47830 | ATG GGA TTA AGA CTA ACA CAA AGC | Wild type Forward | A | Wt F | ||
47831 | CCA TCT CAA CCC AAG ACA TCT AGT | Common | A | Common | ||
47832 | AGT TGT TTT AAA GTA GAT GGA GCC A | Mutant Forward | A | Mut F | ||
47833 | Fluorophore-1 | AGT TTG TGT GAC TGC TCG TTG A | Quencher-1 | WT Probe | ||
48566 | Fluorophore-2 | AGT GTT TCT CAT GCT TTC CAT AAG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47830 | 0.40 uM |
47831 | 0.40 uM |
47832 | 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 |