Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:102681802-102681958 157bp CTGTGGGAGTTAAGATGACTCT CACTGGAGCACTCTAGGCTGA
Mut= 168 bp
Wt= 157 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGTGTTGGGTAAGACTCAAGCTTCTGCCATGCTGATATTCCTGGCTTTGGACCACACTTTCAACAGCAAGTTCTGGGAAATAATAGAGAACAGGACTAATGAGAGGAGAAATAGTCCTGGCTAGGAAAGTCAGGTTATATATCTTTATAGACGCTTGAGGACTGTGGGAGTTAAGATGACTCTCAACCCAGAAAGCctccctgcatgtcacatgggcagaggctgtgccttccgtggcagtccttttcctcttatgttatcccagccagctgcctgaggaacattgggaagcccctgtcagcctagagtgctccagtggcctcctgatggggcacgctcatgatccttctgtctcttgcagtggaaactccagcaggctctggagcatctgaggaaagaacaggaagaagcctggaaactggaagtcagtgagaaggaacaggctgccatctggaaggcaggttgcacttgagaagggggctcactcacatcacccctctcttctagagccgacggtggagtcttcctcacgtctgtgaggcagagtgagggccttcagcctcccttttcctaaggccacataacttctcaggtccagcttatagctttggggtagcagtagcctctgatttcatatcttcacgtatgaggaaggaatggcgccttgcacacgtgagctcctaggagtatagcttatagagtggtgatctaacagtttctgtctcactttcctgagtgacgacggcacagctgctaagactttggttgcctaaatattaggaacccccctcagaaatatatttgaaaccaataggcataaaactctctgaacttttggtccatgtgatctaaaacaatacaataaacataaacacacacaaacacacacaaaacaaagaagaaaaactatccagcagtcttgttagcaaccatcagtgtaaccatctgactggttttctgaaaatgagagctagtccccatcatgccaagataatatcatgtaccaggatggtggtacatgcctagaagtcagccttgggaggcaagggcagagaactgctacagtttcaagtccaacctggtctacatagtgaataacagtccagggtggcaggggctagacactggagccctgtctcaagaagaaagaaagaagataacctagattctgttgcagttgataaaagtagtgcagacagattagaggtcactcatccagacatctgaactgaaagtgctcccaaatccaaagtgtcttatagccaacatgctaccaaaagtagaaggaagtttcgctgaatgataggttatactcagaatgcaagcacactaaaatcattttacaaaattactttcaggttctctgtacaagatacatgtgaagcatggatgaatttcatgttcagagttggatctcattgccaaagtatctctttatattatgtaatctaaaatccagaaaagttccaagtctgaaatactctggtcccactcattatttgtaagaaaatacactacttatgaaaggaccaaaaataatgcctggggccatggggagttaggaacagaaaagttagaccctttgtctttgccttcataacacccagaacatggttgtagcaaagcatatccatcgtgtgtgtgtttgcacatgtcagtgtgctccatcccacgtgacccttaccctaaaaagaccagtgctaattccagtcttgactccagacacagatggaaagacgaaagcagagcatccgatgggagtttgaaaaatatcggcagttattaaaggaaaaggagctgccctgccagcaggcagaagaggaggcagctgcagctcaggctagcctcgagcaggagaagggagagacagcaagcaaactggagttgaggcgcgaggcgatcatccggcagagccaggtcctgtggagcatgattgtggagctggaagagagatcccagagacctgtgcgctggatgctgcaggtgagcaacagccttccgtgggatgcctgagcgagaactggagacggaggctccaggggaaagtattagctagttcttcacggaccctatcatcattctgtccaccagctgaggaccctgtctgccttcagctgacacatgctgctctcatcctgattgggtaaaagacctgacccttaattttctcataggccagaagtaggagtacaccgggtacttactctatagcttggcagcattctagtcccttccatcctggtcatcttgagatggagtctgaaggtgttccatggagccaggtctgctctgctgtcctcccatgagcttctccccacctctaaagtctgtgtaaccctgtgagaaagcccctcccagagtttacgcctgtctagtgtctctgacgcaccgcacattcatgggctgcattttttttctcctagggtattcaggaagccttgaacaggtacgttcccatccctagagactctgtgaaaccctacaggagatagagaggcctggccagagccagggatggagaaggaatgtagactgagtacctacagaaggcagatgctcaccgtCTTACATTAGTGAAAGAAGATTTGTCTCCTTTTTTGCACTTAATTGAATTCATTTCAGATTTTTCCCAATAAGAATTTTAAGTAAAGCTGAAGTATGTTACTTCATGTAAATGGTCCAAAAATTTGATGCGTGAAACTTCTAGCTCAGAAGATCACCCTTTCTCCTTTGTGTCACATCTCTTTCTCTCC
This mutation is a 2368 bp deletion beginning at Chromosome 7 position 102,679,556 bp and ending after 102,681,923 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46838 | CTG TGG GAG TTA AGA TGA CTC T | Common | A | |||
46839 | CAC TGG AGC ACT CTA GGC TGA | Wild type Reverse | A | |||
46840 | CAC GCA TCA AAT TTT TGG AC | Mutant Reverse | A | |||
46841 | Fluorophore-1 | CTG CCT GAG GAA CAT TGG GA | Quencher-1 | WT Probe | ||
46842 | Fluorophore-2 | AAA GAA GAT TTG TCT CCT TTT TTG CAC T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46838 | 0.40 uM |
46839 | 0.40 uM |
46840 | 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 |