Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:124583670+124583782 113bp TTCTTTGCCATGGGTTTAGG GGCAGTCTGGTGTCAAGTAGC
Mut = 119 bp
Wt = 113 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CATGTGGCCCCACCATGTGTATATGATAGGTAAGGTTCTAATCTGTAAATGACTCTGTCTGTGAGGGGAAGAAGgtctcctctcagcttctttgccatgggtttaggaaaagaaaagctgagcagatttttagcagggaatccttgtcagatgtcattgagcatctgacaggactcgctgctacttgacaccagactgcctggggccacgtagcaagaccctttcttacacacaccacagtgtataataagtaaataatgctaagtaatagctactttttctttcaacgactcctccctcgaggctgatggggttgtgtgggagctgacatgttcctctcgtgtctgtcagtttactttatccaaatgcatggatgcagtgatggtgctggcgaattctcacctgttcatgaaccgctccaaccagctggctgtcatcgccagtcacattcaggaaaggtaggggccagaggctgcttttcagtcctttaacagttgcatgcatgggtagagcaccttccggttactcagaggatctttctcctccccctcagccggctcttatacccggggaagaacggtggacttggagacttcttcggagaccctggcaatgcgctccccgactgtaacccctctgggagtaaagatgggaaatacgagctgttgacagttgcaaacgaggtgatcgctgaggagatcaaggatctgatgaccaagagtaagagcctggctattgtcactgctgtctccatgtggtgggaattggtGTATGTTTTTAAAGTGCAAGATTCTTATGAATTCTTTAAGAATTTCATACATGTATGCCTTGTGTTTTGATGATATTCACCAATCCCATTCTTCTCCCTAACCCCAGATCCACTTTCATCTCCTCCCTACATCTCCTCTCCTCCCTACATCTCCT
This mutation is a 695 bp deletion beginning at Chromosome 5 position 124,583,657 bp and ending after 124,584,351 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51208 | TTC TTT GCC ATG GGT TTA GG | Wild type Forward | A | |||
51209 | GGC AGT CTG GTG TCA AGT AGC | Wild type Reverse | A | |||
51210 | CTC TGT CTG TGA GGG GAA GA | Mutant Forward | A | |||
51211 | GGA GAA GAA TGG GAT TGG TG | Mutant Reverse | A | |||
51212 | Fluorophore-1 | CAG GGA ATC CTT GTC AGA TGT CA | Quencher-1 | WT Probe | ||
51213 | Fluorophore-2 | CAT ACA TGT ATG CCT TGT GTT TTG ATG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
51208 | 0.50 uM |
51209 | 0.50 uM |
51210 | 0.50 uM |
51211 | 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 |