Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:72471121-72471217 97bp TGGGACAGTGAGATCAAGCA AGGTTGCCCTCCAGAGAGAG
Mut= 98 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (CT insertion):
ATTAGGATCCAGTGGCTTGGGACAGACACACCTTGGGAGAGGTGCCACATCTCCAGCCCTGGTCATAGCTTGCAGTGCATAGTAGCTTGGTCAGGCCTGCATGAGAAGGGAAGATGGAAGGGTCAGCCTGTCTTGGGACAGTGAGATCAAGCAC^aacagggaagcttgggcaggatggccgtgtgcggtggacagtggatccatcaggtcctctctctggagggcaacctgatggatcaccaaaaggaacctggggcaccccctgagctcacaaggcccatctctttcctgctagtaatctgcaagcagcaggccccagagctggagcccacctcagccatgccccctctgccacaacctccgctggctcccacagcgtccctcactccggcccagggcaccccatcaatggacgagctcatccaacagagccagtggagtctccagcagcaggaacagcacctgctggccctgcgccaggtatgcctgcatgtactgtcagtgaccctggagtcaccttccagtaggtctgtgggtcatccacaggcatcgctcagtggacttgtacccattgtggatgccaacacatggcggcttaggtacctaccattcttaatgagtagggaggggcattattggccccatcttttaggttggttctgttgccaaattctatagg^GATGAGCTACCTGAAATGAAAGGAGAGAAGACATACAGACCTGAGTGCCAGCCCCACTCAGGGTTTCCTTGCTTACCACCATCACATATCATGCATGGATGTTTACCATCAGTCATAGTCTGGACAGTTAGGTCTTAGACAGCAGAACCAGCCTCTACTCTTGGTGACCTATAC
This mutation is a 525 bp deletion beginning at Chromosome 8 position 72,470,672 bp and ending after 72,471,196 bp (GRCm38/mm10). In addition, there is a 2 bp insertion (CT) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39949 | TGG GAC AGT GAG ATC AAG CA | Common | A | |||
39950 | AGG TTG CCC TCC AGA GAG AG | Wild type Reverse | A | |||
39951 | TAA GCA AGG AAA CCC TGA GTG | Mutant Reverse | A | |||
39952 | Fluorophore-1 | ACA GGG AAG CTT GGG CA | Quencher-1 | WT Probe | ||
39953 | Fluorophore-2 | CCT GAT GAG CTA CCT GAA ATG AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39949 | 0.40 uM |
39950 | 0.40 uM |
39951 | 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 |