Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 129 bp
Wild Type = 127 bp
>chr11:107734729-107734855 127bp GCATGACTTCTTCCAACAGG TCACACAAACAGCCACCTTG |
Wt Sequence (deletions in lower case):
TTCTTCCTGTTGTGAGTCTAACCTCCATCTCTCTCCCTGCCCCGCTTTCTTTCAGGCCTCAgtaatatcatcggtatcatcgtctacatttccagcaacacgggcgaccccagtgacaaacgtgacgaagacaaaaagaaccattacaactacggctggtctttttactttggagccctgtcgtttattgtggcggagaccgtgggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaagcgggagttccttaaggcctcttcctcctctccttacgccaggatgccgagctacaggtaccggcgacggcggtccaggtccagttcaaggtccacggaggcctcaccctccagggatgcatctcccgtgggcctgaagatcaccggagccattcccatgggtgagctgtccatgtacacgctatccagagaaccccttaaggtgaccacagctgcgagctacagtccggaccaggacgctggcttcctgcagatgcatgacttcttccaacaggacctaaaggaaggtttccatgtcagcatgctgaaccggcGGACGACTCCCGTGTGACCCGCCCACCCCTCTCGGCACAGGCCTCCCCCAAGGTGGCTGTTTGTGTGA
This mutation is a 516 bp deletion beginning at Chromosome 11 position 107,734,797 bp and ending after 107,735,312 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64895 | GCA TGA CTT CTT CCA ACA GG | Wild type Forward | A | |||
64896 | TCA CAC AAA CAG CCA CCT TG | Common | A | |||
64898 | Fluorophore-1 | TTC CAT GTC AGC ATG CTG A | Quencher-1 | WT Probe | ||
66312 | TTC TTC CTG TTG TGA GTC TAA CC | Mutant Forward | A | |||
66313 | Fluorophore-2 | TCA GGC CTC AGG ACG ACT 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 |
64895 | 0.50 uM |
64896 | 0.50 uM |
66312 | 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 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |