Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:31064317+31064437 121bp AAAACAGCACATGCTTATCCAC AAGCAGGATTGCAAAGCACA
Mut= 123 bp
Wt= 121 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case and insertion with carrots ^g^ (ACTGTAGGAACTGATGAGAGTTG insertion):
TGGACTGGGAAGAGAAAATTCCTATATGAGTAGTTTTAGACACTTTTTGTGGGAGTTGAGAATTTATTCTACAAGAAGACAGTTGTCACAGGTTTTAAAGGGTGAAATAAGGGTCTAAGTTAAAGCAGAAGAAAGGAGGGAATTAAAGAGGTGAAGGGCATCTAAAAAGC^agcctgggatctgttttgtgttttatgtccacactaaagatgctggacatttagacaattacattgctttatacatttttctaagtgaaaatctgtttttaaaagtctttgttgcttcctttgttcatttaagagttggactacagaaatgtgtattcaacttaaacatcagagcctcgttaccttctttggtttatttttttagattggaaaatgttcagtatccctatcaactctacattgctccttctaccagcagtacagagcggccaagtccaaatggtccagacagaccgtttcagtgtccaacctgtggggtccgcttcacccgtattcagaatctaaaacagcacatgcttatccactcaggtaatgtcaccttcagggttgttgtatgggggggg^GGGGCGGCACGGGGGGTTGCAGGCAGGCAGGCAAAAACGTTGTGCTTTGCAATCCTGCTTAAAATACAAAAGACTTTGTGCCAGGCCAGACATAGTGCCAAGTGCCTTTAATCCCACCACTCAAGTCTAAGGCAGGCAGATCTAGAATCTGAGGCCAGCC
This mutation is a 404 bp deletion beginning at Chromosome 9 position 31,063,974 bp and ending after 31,064,377 bp (GRCm38/mm10). In addition, there is a 23 bp insertion (ACTGTAGGAACTGATGAGAGTTG) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44535 | AAA ACA GCA CAT GCT TAT CCA C | Wild type Forward | A | |||
44536 | AAG CAG GAT TGC AAA GCA CA | Common | A | |||
44537 | GAA AGG AGG GAA TTA AAG AGG TG | Mutant Forward | A | |||
44538 | Fluorophore-1 | ATG TCA CCT TCA GGG TTG TTG TA | Quencher-1 | WT Probe | ||
44539 | Fluorophore-2 | CTC ATC AGT TCC TAC AGT GGG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44535 | 0.40 uM |
44536 | 0.40 uM |
44537 | 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 |