Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant = 94 bp
Heterozygote = 93 bp and 94 bp
Wild type = 93 bp
>chr3:98061028+98061120 93bp ATGTGGACCCTTGCATCTTG CCTATGTGCCAAACACAGTGAC
This assay is capable of distinguishing hemi from hom. Transgene insertion site is known to be on mouse Chr 3.
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
This protocol can be used for conventional PCR. Omit probes and run as a separated assay.
Wt Sequence:
AATTGAAGTCTGGAGCCTTTAAGTTGTAAAGATTATGTGGACCCTTGCATCTTGCCCTTTGGCATCCGTTAAGAATTTGAACAGCTGAATGGCTTCCATATGCTAGTCACTGTGTTTGGCACATAGGGTATAGTAGAGAATAACTCTTGTCTATTAGAGAAGATAAATAATTGTAGTA
Mutant Sequence:
CCCAGTGGTTCCCAAACTTCCAGTGTAATTGAAGTCTGGAGCCTTTAAGTTGTAAAGATTATGTGGACCCTTGCATCTTGCCCTTTGGCTGCATTTTTCCCCCATAAACCTTCTGCTGATGTATCAGGTTGCATTTTTCAGTGTGATGACTCCAATTTCATTCCCTTAAGCCTTCATCCATGAGTTACCAGTATTACTCTGTGTAAAGGAAAAGTGAATTGCACCTGTT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
55827 | ATG TGG ACC CTT GCA TCT TG | Common | A | |||
55828 | CCT ATG TGC CAA ACA CAG TGA C | Wild type Reverse | A | |||
55829 | Fluorophore-1 | CAT CCG TTA AGA ATT TGA ACA GCT | Quencher-1 | WT Probe | ||
55830 | TGG AGT CAT CAC ACT GAA AAA TG | Mutant Reverse | A | |||
55831 | Fluorophore-2 | CTG CAT TTT TCC CCC ATA AAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
55827 | 0.40 uM |
55828 | 0.40 uM |
55830 | 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 |