Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:79736759-79736864 106bp GTACTGGGCACAGGCGTTAG AACTCTTCTGAACGGCTTGTAA
Mut= 112 bp
Wt= 106 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (AATCATCTCA insertion):
CGCCGTGCTTTTCACTGGCTCTGTAGAGGCTTGAATGCTGTGTGTGTGAGGTAATGAGGTAAAAGTTGATGTGTGTCGGGAGTCAGGCCACTGATGGAGATATTTAAAGATGCTACACGTGTCTGCGAGTACTGGGCACAGGCGTTAGCTCTCACCCAT^tgtgtgtgctgatttatagtatcagatgatgatcagttctgtccagtgcactgttacaagccgttcagaagagtttaaataaagataaaatgaccaagcatctctctttctctctttctgtctccacagaaccatccgagtatggctgaaaagagacagcggccagtactggcccagcatctaccacacaatggcctgtaagtcctgggctttcccacttaagaccttgtttctgataccttggatgattgatatttttaaaagacctggctcaaaattgccactgaatttcagagtgagtgcctttgttgcccaagacctctgg^CAGGCTATCATAAACCACAGGAAACTCCCTTTGCAGAGGCAATTGGTCTACCAGCCTAACTGAAACAGGACCAAAACTGAAAAAGGACTAAAACAAAAAAGGAAAAGGACCATTTTCCTTTGTCCTAGTTTGTCCAGTTTAAACTCAGTAGTGACTTTATGAAAATGGT
This mutation is a 325 bp deletion beginning at Chromosome 1 position 79,739,934 bp and ending after 79,740,258 bp (GRCm38/mm10). In addition, there is a 10 bp (AATCATCTCA) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46149 | GTA CTG GGC ACA GGC GTT AG | Common | A | |||
46150 | AAC TCT TCT GAA CGG CTT GTA A | Wild type Reverse | A | |||
46151 | GTC CTG TTT CAG TTA GGC TGG T | Mutant Reverse | A | |||
46152 | Fluorophore-1 | ATG ATC AGT TCT GTC CAG TGC A | Quencher-1 | WT Probe | ||
46153 | Fluorophore-2 | CAT CTC ACA GGC TAT CAT AAA CCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46149 | 0.40 uM |
46150 | 0.40 uM |
46151 | 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 |