Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:113639527+113639616 90bp AGGCTCTCCTTTCCAGTTTAATG AAGTTATGGGGCTCAGCTTC
Mut= 86 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTTAGAGCCATGGGTACACCTACCCACCCACCAGAAGTGAGTCAGTGGGTCAGGGTAACTGTCTGGAGGTCCTTCACATTGTCTGGGAGTGAGGAGGTGGACTGGATGACCTGGCTATGTCTGAATCCATGTTTGTCTCACCATCCCAGCACccgaccccgtgctgtgtctaagttaccagatacctccacagccaacatatccccacaagtgtcccaataaccccagcttgtcctcgccctaatttaggtgctggcagagtaagagcgcaggctcggtcaggccaggcgctgtatgtgttggagagggtttgggaaaggcgtcctttctgaccccttggctcattcctggtctgtgtccaccctctccactcttccttcccccagaggatcacaagctggtgttcctgcagcagggtccactcctgctcgtggctgtgtcccggactccacagtctgcagcacagctacgaggtgaactgcttgctgtacacgcccaaattgtgagcacactaacgcgtgcaagcgtggcccgcatcttcgcacataagcagaactatgacctccgtcgcctgttggccggctcagagcgcacactggaccggctcttggacagtgtggagcaagacccgggcgccctgctcctgggcgctgtgcgctgtgtgcctctggcccgtccactgcgggatgccctgggcacactactgcggcgctgtacagccccaggcctggccttgtcagtgctggctgttggcggtcgactgataaccgtggcccaggagaggaatgtgctggcggagtgccggctggaccctgctgatctacagttgctgcttgactgggtgggtgcaccagccttcgcggcaggtgaagcatgggcacctgtgtgcctgcctcgctttaacccagatggtttcttctatgcctatgtggcccgcctggattccatgcccgtctgcctgctgctgcttggtaccaaccgtgaagccttccatgccatggccgcctgccggcgcttagttgaagatgggatgcacaaccttggtgccctgcgtactcttggggaggctgccaacttctctaatggcccagcagccagtgccccagcctacagtgtgcaggctgtgggagcccctggcctccggcattttctctataagccactggatatccctgaacaacaccgccagctgccacagtttaccaggtgggccctgaccccaagttgattgaagatatgtctgaccaagacagtcagcatcttcttcacaggagttaggagagatagtgaggccatgctctagaaatggggagcagacagttctctgatggcagaggcttgagcaccaacctacaggagtccccctgttgtgggagaaaggctctcctttccagtttaatggaggcagcttgtcttacaaccacgagatacCCTCAAGTCTAATGGGAGAAGCTGAGCCCCATAACTTAATCACAGTCCCTAGCCTGAGAGGGGAGGCAGGCTCTGACTCACCATGCCACTGCCTCCCTCTGTTGCAGTCCCGAGCTAGAGGCCCCATACA
This mutation is a 1279 bp deletion beginning at Chromosome 8 position 113,638,301 bp and ending after 113,639,579 bp bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43733 | AGG CTC TCC TTT CCA GTT TAA TG | Wild type Forward | A | |||
43734 | AAG TTA TGG GGC TCA GCT TC | Common | A | |||
43735 | GGA TGA CCT GGC TAT GTC TGA | Mutant Forward | A | |||
43736 | Fluorophore-1 | AGG CAG CTT GTC TTA CAA CCA C | Quencher-1 | WT Probe | ||
43737 | Fluorophore-2 | CAC CAT CCC AGC ACC CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43733 | 0.40 uM |
43734 | 0.40 uM |
43735 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
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 |