Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:53390818+53390951 134bp TTCAGGGCTCAGAAATGTGT CTCGTATTCTAGCTTATTCTGAAGC
Mut= 136 bp
Wt= 134 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTTACAGGGAGAAAGTTGAATTGCCACAGGGCCATCATCTTTAGAATGTGGACTGTTACTGCTTGAATGTTATTCAGAGGAGAAAAGACCAAGTAGGAGAAATGTTACTTTCTTCAATGAATTAATTTTCTCATCCACTGTTATTGAATGACTGATGAgtcccatgttaagtcagatccttgccttaaggatggaccacacatagttgctgtctgcaaggatcttccaggctgaaatgagcatggtttaccatgaacactgcaggacaaaggactttcactgagacataatgatcaggaaatggcaacgttatttctatttcgtttgcttacaggtcaaacacagttcaaagtagtggtaaaatccctttcacctaaagagctggtccgtatttatgtccctaaacctttggacagaaatgatggaacgtttttggtgagatacaggatgcacgagacggcccataaagggctcaagatagagatccttcatggcagtgaacatgtggctcactctccctatattttaaaaggtacgtgttactactcacctgaccctaggcatatatgggatacttgcttttcagaagggctgtaatgaccctgcagtcaaatgtgacagagtaaacattgtatatgtacttctggaagttcaaattttacttcattttttggcatttaaaaaatgatcattttctgagtttggagagcccttgctgctcttaccgagggcctgggttcagttctcagcaccacatggtggctcacaagtcagctgtaactccagttctaggggatccaatgtccctttctgaccagacaggtatacatgtgacatgcacatacatgcatgcagacaaaacacacacataaaacaaagaaacctcttgaaaaatcctttcctgtttcattttgtatagttcctattgctgtatctctaagtttgttcatcttttgtcctgcactgccagagttgccattaatcccctccagtgtaactttttaattacactggtaacatttttaattttcctaagctttctctctgggatgtggcagagttaattggtcataatctgaccttttcctgttatatttttacagtgtgttagatgaaattgagtcatgtttaatctggggctaattactctcagcaactgaggtgcacacaccccaagtcccaagaatgaggaggctttccagccaggctgacacaagcacttggcccagccctgcaggagcgctatgtactggacacaagcacttgacccagccttgcaggagcgctatgcactggccaccagcgttgctctgatggtcttccctgcactgggtggattccactcacagatgccctggttacaggctgggctggactctccttctcagagtcctgaaggcttttaggctttcttgtgtttagttctcacccctcatacactctgttcccaggctcagctgcctgggtctctggcatgctcagctcttccttaacacagtgagactgctcagtcctgcacacactctcttcctgagctacagtaagcagcaagctgtgtgacctgtgaggtttcccttgctagtgtcacctccaagacccactgtccttccctctctcgtgtccataatctagaaactgtgagtccacatgacaggtttgaagtaaaagtcagcgcctcttcccagtctgtgtctgaagtagaaaactccagcaaaatgaggcctaacgtcatctcagcactggggaagtagagacaggaggatctctgtgtgagcaagcttaagactagcctgggctatataagactctgtttccaaagatcacaaataaaccatcaagaaaatatattcagttaaatacatgtgctgaaattatgttagatttgtatatattgtttctaaatgtgtcttcagaaaagtcttcagaaaagttcagctgtccacttacactgatagacccctctgggttactctttcctctgagatgtgctgaccaagggggcattagggaccttgagggaaaggccctccattttctcaaaaacttaactatttctgcatttgaatgtctgaaaatcaagaatttttctgtgctaaaatggagtcactttgcatttattacttatacctcagcagaagtggaggagtcatccacgtgactggagtgtgctagtttggaagtgagcatgtgctctgtagtctgtccagggggcctagttgggggcagaggtggggctctcacatgatcactctactacgaattgacctaagataggttcacctctgtcaggagtttccagatctgccaagctgccatccggtgctcacttcttcataggtcatggtgtttgttaactcagggtgacactgtgtgtgtgttccctccttaggacccgtgtaccacgagtactgtgactgcccggaagatgaccctcaggtctggcaggaaaccctgtcttgtccagccagtgaaccacagatcgagcaggattttgtttccttccccagcattaatctccagcagatgctgaaggaggtccccacaagatttggggacgagcggggtgctgttgttcattacacaattctcaataaccacatctaccggagatctttggggaaatacacagacttcaaaatgttctctgatgaaattttgctgtcactggcaagaaaggtagggaaacaaatgaactgcttccagaaatgatatcattgtgttgttgtggcaacagttacatacttaggacgataaagactggtgagaacagatttcagggctcagaaatgtgttccagaacgagcgtgagtctcctacctttgcagatggctttgctgtaagatttaggttgaGGTGGAATCCCAGAACTCAGGTTCATAAGGCTTCAGAATAAGCTAGAATACGAGAGTGAGGCGGAACGCCAGGACAGAGCACAGGTCTTGTTTAGAAGTCTAAAGGTTCTGGAGAGCTCAGTCAGGAGATTAATGTGGGGGGTAGAGATTTGGGCAGCT
This mutation is a 2694 bp deletion beginning at Chromosome 9 position 53,388,204 bp and ending after 53,390,897 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45410 | TTC AGG GCT CAG AAA TGT GT | Wild type Forward | A | |||
45411 | CTC GTA TTC TAG CTT ATT CTG AAG C | Common | A | |||
45412 | GAG GAG AAA AGA CCA AGT AGG AGA | Mutant Forward | A | |||
45413 | Fluorophore-1 | CTC CTA CCT TTG CAG ATG GCT | Quencher-1 | WT Probe | ||
45414 | Fluorophore-2 | CAC TGT TAT TGA ATG ACT GAT GAG GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
45410 | 0.50 uM |
45411 | 0.50 uM |
45412 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
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 |