Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:56241294-56241420 127bp CACACCCTGCTCTATAAATC GTTCTTGGATCCCGAGGAGA
Mut= 126 bp
Wt= 127 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTCTTCCTTTAAAAAAGAAAAACAAGCAAACATGTAGGTTTAATGACCAGGTATGAAGGTCTCAGATCCTTTCTAGTTAGCTCTGACTATCCTGGAACTTACTATATAAACCAAGCTGGCCTGGAACTCACCAAGCTATACACCTGCATCTGCCTCCCAAGTGCTGGGATTAAAGACCTGTGCCACCACACCCTGCTCTATAAATCTAAATGTTAATAGAACTTTTGAATACAAATTTgatggactgtcttatttgtttcagtcgtgtgacccatgaagtggcaggagaactttctcctcgggatccaagaactcctgctgggaagcaagatggcacttcagtcacaccaacactgcctccacctgacctggaaagggaagaagaaaaagatgacactttggatcctactgacgtgagtccctgcagtgcaacctacagcaacctaggtaagtacaaaggttatatagatagtacctaagccgcgtctTTATGTACAGTAACACCATTAGAGTTATAAATCTACATTTGTTTCAAAACATAACATTCAGGTTTTACTAGGCTTTTTTTGGGTTTTTTTGTTTGTTTGTTTGTTTGTTTGTTTTTTGTTTTTTTGGTTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCTGTCCTGGAACTCACTTTGTAGACCAGGCTGGCCTTGAACTCAGAAATCTGCCTGCCTCTGCCTCCTGAGT
This mutation is a 250 bp deletion beginning at Chromosome 9 position 56,241,119 bp and ending after 56,241,368 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47678 | CAC ACC CTG CTC TAT AAA TC | Common | A | Common | ||
47679 | GTT CTT GGA TCC CGA GGA GA | Wild type Reverse | A | Wt R | ||
47680 | AGC CTA GTA AAA CCT GAA TGT TAT G | Mutant Reverse | A | Mut R | ||
47681 | Fluorophore-1 | CAG TCG TGT GAC CCA TGA A | Quencher-1 | WT Probe | ||
47682 | Fluorophore-2 | CAA ATT TTT ATG TAC AGT AAC ACC ATT AGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47678 | 0.40 uM |
47679 | 0.40 uM |
47680 | 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 |