Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:181590356-181590458 103bp GAGGGTTAGAGTGGGGTCCA AGGGTACATGCTCAAACAAACT
Mut= 112 bp
Wt= 103 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (CCAGCCCTCAC insertion):
GACCCACCAAAGCTAGGTGAGGAGCTAAGGCAGAAATCAGTTCTAGAGGATACAGATAGCCAGTGGTAGAGTTACCTCCTTGGGTATCCTGAGGACCTTTGTGGGGTGGAGATTGTCAGCCTTATTGTAGCCATGCTCTTGTCCTGGGTTAACTCCAGCCACTGGGCCTTTATCTGGGTGAGCCAAGACT^cagtctgggccaccgcccttgggggtcggtgatattgggcaaccactgagccttactgtctcctgtactatagggatgccagtggtccacagtggacatacagtgcctagccaggccccactctgctttgaccctggaaactcagccagtgacaagacagaagggaagaaaaaagggaggccgaaagccgagaaccaagctctccgagatattcctgtgagctccctgagggttagagtggggtccattcaatttagtctgtgaaggccacttccatgcccttaccagccctcca^TATATCCTGTGCCAGTTTGTTTGAGCATGTACCCTAGGGCCCTTGTGTCTGTTACAAAGTCCCAGTGGGCTGTGAGTAGTCATCAGCATAGGTCTGCAGCATGGCTTGAATTCCTGGTCTGCTCTTGAGGTTGAAGGGCTTATTTGGCTTGGGGGAACTTTAAGTCTGGGTAGCTACAGGTCAGCAGCTTGGCCTTTTCCTC
This mutation is a 295 bp deletion beginning at Chromosome 2 position 181,590,391 bp and ending after 181,590,685 bp (GRCm38/mm10). In addition, there is an 11 bp insertion (CCAGCCCTCAC) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47476 | GAG GGT TAG AGT GGG GTC CA | Wild type Forward | A | |||
47477 | AGG GTA CAT GCT CAA ACA AAC T | Common | A | |||
47478 | TTG TAG CCA TGC TCT TGT CC | Mutant Forward | A | |||
47479 | Fluorophore-1 | TGT GAA GGC CAC TTC CAT G | Quencher-1 | WT Probe | ||
47480 | Fluorophore-2 | CCA AGA CTG TGA GGG CTG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47476 | 0.40 uM |
47477 | 0.40 uM |
47478 | 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 |