Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:114425857+114425996 140bp CTCCATATCCTCCCAAGTCA ACATGGTCTGTCTCTGCATACC
Mut = 150 bp
Wt = 140 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTAAAAGTCCAAAGGAAACCCACCATGTAGTTCCTTACATAAACTAGGCTACAAATTAGATGGAAGCAGCCCAGGGTTTAAGATTACATTTTGAGTCATATGTGGCTGTGAGTTCATTATATGTCTCCTAATCCCTTtggggggcatttcaagttgtgagtgacaaattctctaagcctcaaaaacctgataaatcgggatagcaccaccatgtcttggggttgctggagttccgtgtagttattgttgtgtctggcaccaacaaaaccatcaatggtaactcttcactgtcaacccactaatcattacacaaatgtttaccaatgcttgccaggtcccgtattaggcatatcacacacaaaaggcaccgaatgacatcaccaactttcccatcttcacagtctgtctccgaggcaccaaagttcataagaagtgctaccttgcttcagaaggcctcaagcattaccacgaagccaacgaagactgcatttccaagggagggaccctggttgtccccaggaattccgacgaaatcaatgcccttcgagactatggtaagaggagtctgccaggtgtcaatgacttttggctaggcataaatgacatggtcacagaaggcaagttcctcgatgtccacggattcgctgtctccttcctcaactgggaccgtgcccagccaagtggtggcaagcgggaaaactgtgtcctgttctcccagtcagctcaggggaaatggagcgacgaagcctgtcgcagtagcaagcgatacatatgtgagtttatcatcccataagaagcccctcctccatatcctcccaagtcagactggtcagccatgataaataagctcgtaggccccctcagtaaACTCGGATTACATTTATTCATTGCAGCATCACAATGCCAGGATTGGTAGTCATAGGTATGCAGAGACAGACCATGTTTCTACCATAGCTCCTTGTAATATTACTCTTCGCATGACAAAGGGGATCAGAAAATAATGACCCAGACATGGTAAGAAAGGGACTTCTGATAATGGGCAGTCAAATTGTCTCCC
This mutation is a 730 bp deletion beginning at Chromosome 8 position 114,425,191 bp and ending after 114,425,920 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50709 | ACA TGG TCT GTC TCT GCA TAC C | Common | A | |||
50710 | CTC CAT ATC CTC CCA AGT CA | Wild type Forward | A | |||
50711 | AAG CAG CCC AGG GTT TAA GA | Mutant Forward | A | |||
50712 | Fluorophore-1 | AGC CAT GAT AAA TAA GCT CGT AGG C | Quencher-1 | WT Probe | ||
50713 | Fluorophore-2 | CAT TAT ATG TCT CCT AAT CCC TTA CTC GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50709 | 0.40 uM |
50710 | 0.40 uM |
50711 | 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 |