Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:142440520+142440605 86bp TCTCTGCTCAAGGCCTGATTC TCCTGCTCCTCTACCAGTCC
Mut= 95 bp
Wt= 86 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGGGGGTGGGGGGGTGCAAGGTTCAAGCTGGGGAGCTCAAGGCGGCCCTGGCAGGCCTTCTTCTCAGAGGAAGGAGATGAGCAGAGGTGATAGGGAACGAGTGATTCAGGAAGCAGGCAGATGTTGGTCCATGGTCcattggaagctacagtagtagcagtagtagcatatatgtcacattgaaggaagattggctggtctcagggagctgatctgtgttacaatagtccagatctctggttaaagattggtctcaatcatacttgcctgatctcccaccatccaccccaggttcaaccagatgtggactgcctagaaccctgctctggaggggaccaacagtggggtcgactcctgctgtcactagagtatgattttggaagccaggaggtaaggctcactcagcaggtccagaggctctgtggttgatggagaatgtgatgaatgaagagtcaggtagaggggagcaggggaaaagaacctcttgaccccaggctgagccaacccactggccctcagattagagtgggcctgaggcaggctggaaacctgaaggctgagggcacagcagacccctacgcctgggtcagtgtgtccacccagtctgggagaagacatgagacaaaggtgcaccgagggacgctctctcccatgtttgaagagacatgctgctttctggtaagtctagtctgggtagatagagtgggctggagccagggcagcaggacccagctcattaccctgtctgcaggtcccaccagcagagctgcccaaagccactctgaaggtgcagctgtgggacttcaagcggttttcagaacacgagccactgggtgagctccaactacccctgggcaccgtggaccttcaacatgtcctggagagctggtaccagctaggccctccagggaccactgaggtgagcctggctggtaggggcccgggatctacctgtaacatggggggaggacaaggtctgaatgctgtacctgctgagttcaaacccccaagggtaaggatgaccctcccagttgcagccccgtcttgttgcccatagcctgagcagatgggggagctgtgcttctcactacgctacgtgcccagctcaggcagcctgactgtggttgtactggaggctcggggtctgaatccggggcttgcaggtgagagctatacttttgtggacagcgagctggaccctcgtgtgcatgtatgggactcccttcttgaactttctcactcgtgcagaggcctacgtgaagatccagcttatgttaaaccagagaaaatggaagaagagcaaaacatcctccaagaaaggcacgactaccccctacttcaacgaggccttcgtcttcctggttcccgttagccagctccaggtagggagctgggaggaagggcagagtggcctgggacgttgtgccatgccaaactgacaaccatcccctctacatgtctgcagagtgtggacctggtgctggctgtctgggcccgtggtctgcagctccggactgagcctgtgggcaaagtgctgctgggttcccgggcttctggccaacccctacagcactgggcagacatgctggcccatgctcggcggcccatcgcccagtggcaccacctgcggtcccccagggaagtggatcgtgttctggccctgcagcctcgccttcccctgcttaggcctcgctcctgaaagcaccgcgggtctcagttttcccagatggagagctgtggatatttgtgcaggctcttcagatctctctgtaataaacaaattctctttcacactcacatgtcctgatggagccctgtgaggaggatctgagatacaaggcaggctacactttggcaccactgtcctctcaaggttggggtagggtgctctgaacagatttgccagtcctgggcctctctctgctcaaggcctgattcctaggcctttccctgaaaaacgggcacCACCCTCTCTGCCAGCCAGGACTGGTAGAGGAGCAGGAGGCTAGAGCTGGCTCTGTGAAGGCAAAGAGGGCAATTGAGGGGGAGTTGTGGGTAGGTACAGCTTGTGGAAAAGACCTCAAGACCATGAGGGACTGTGATACAGTG
This mutation is a 1829 bp deletion beginning at Chromosome 7 position 142,438,739 bp and ending after 142,440,567 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44709 | TCT CTG CTC AAG GCC TGA TTC | Wild type Forward | A | |||
44710 | TCC TGC TCC TCT ACC AGT CC | Common | A | |||
44711 | AGC AGA GGT GAT AGG GAA CG | Mutant Forward | A | |||
44712 | Fluorophore-1 | TCC CTG AAA AAC GGG CA | Quencher-1 | WT Probe | ||
44713 | Fluorophore-2 | CAG ATG TTG GTC CAT GGT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
44709 | 0.50 uM |
44710 | 0.50 uM |
44711 | 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 |