Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:3933373+3933515 143bp GTTGAGCAACACCACAGCA GATCTTCCCTGGCCTCTACA
Mut= 140 bp
Wt= 143 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCCTCCCTCCCTCCTCTCTCTTTCTGAAGATCAAACTCAGACCTGCATGCTTGTAAGGCAAGCACTTTACTGATGGAGCCTTTGCCCTAGCCTCCTTTCTGAGTCCTTTGTTCTTGGGAAGCTATGTTCTACCATTATCATGGTAAACAAACAAGAAGGCACTTTCAGAGTTTGGATATGAGTATTCTTCAAGTAAGAAAGTACTGATTCCTTCTAGTTAACTGACTGTAATTCTCACTCCCCCCCCCCCGCCCCCAGgcagaacaatgcagtggacttcagggatttttgatttaccgaagctttggaggaggcacagggtcgggatttacatctctcttgatggagcgactctccgtagagtattgcaagaagataaaattggagttctctgtctacccatctcctaggatctccactgccgtggtagaaccgtacaatgccatcctcaccactcactccaccatagaatactcagattgtgctttcatggtggacaatgaggctctgtacgacatctgccagcacaaacttggcattgaacgtccttcctatgctagtatcaacaggctgattgctcaggtgtcgtcttccatcactgcatcactgcgttttgaaggacctttgaatgtagacttaattgaattccagaccaacctagtaccttatccaagaatccatttccccatcacagctcttgcccccatcatctccgctgaaaaggcgtaccaagagcagctctctgtgtctgacgtcactgcttcttgctttgaggtctccaaccagttggtcaagtgtgatcccaggcttgggaagtacatggcttgctgcttactgtacagaggagatgtggtccctaaggatgtaaatgaagcaattgcagccatgaaatctaggacctctgttcagtttgttgattggtgtccaactggtttcaaggtgggcatcaattatcagccacctgcagtggtaccaggaggggatctagctagggttcagcgtgctgtgtgcatgttgagcaacaccacagcaattgtggaagcctgggcccgcctggaccacaaatttgacctcatgtatgccaagaaggcattcctgcactggtacatcacagaagGCATGGAGCTTGGGGAGTTTGTAGAGGCCAGGGAAGATCTGGCTGCTCTGGAAAAAGATTATGAAGAAGTGGGTCTGAGTTTCTGACTCTTTGTAAGTCTGGTTTTTCCTTTATTCATGTGTCTATGTGAGGTGTGTGTGCTTATTTGCATATGTGCACTCATATGTGGGGAAGCACGTGTATGCACAAATGTATGTGCCCATGTGTGCACAAGTGTGTGGAGGGCAGATGCTGAACTGGGTGTGTGCCTTCCTT
This mutation is a 859 bp deletion beginning at Chromosome 13 position 3,932,618 bp and ending after 3,933,476 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49005 | GTT GAG CAA CAC CAC AGC A | Wild type Forward | A | |||
49006 | GAT CTT CCC TGG CCT CTA CA | Common | A | |||
49007 | GGC ACT TTC AGA GTT TGG ATA TG | Mutant Forward | A | |||
49008 | Fluorophore-1 | CCT GGA CCA CAA ATT TGA CCT | Quencher-1 | WT Probe | ||
49009 | Fluorophore-2 | CTG ACT GTA ATT CTC ACT CCC CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49005 | 0.40 uM |
49006 | 0.40 uM |
49007 | 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 |