Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 107 bp
Wild Type = 141 bp
>chr4:152280454+152280594 141bp TCGACAGCTAGGACCTTTTTG TGACCTTCTCCGGTCCTTAG
Wt Sequence (deleted region in lower case):
ATTCTACACCCTCACACTGGCCACCTGCTTCTCTGTCACCTCCATCTCCTACCACCGCATGTGGATGGTCCGATGGCCAGTCAACTACCGGTGAGCGCAGGACAGCCTGGTGTTCTGAGGGAGGGGCTGTGcttgaggcaaaggcatgcctggtgtacctgtatgagggctccagaatgggtgatggggttgagggctggcatagtctggggctactggggggggggtgtgcgtgttgaggtagttatggcagaatgttagtgtagcttggtacacaagtgcctcgtgagagggacagggacacgaatagcttttgagtgaggtacaaggctctgccatgatggctgggtggaggctacagtgtcagtggggagggatggtctgggccctctgtggtcccatagaggcctgtgagtaacaccaggattctctattctgccctgctccctgccaggctgagcaacgccaagaagcaggcggtgcacacagttatgggcatctggatggtgtccttcatcctgtcggcccttcctgccgttggctggcacgacacgagcgagcgtttctacacccacggctgccgcttcatcgtggccgagattggcctgggcttcggcgtctgcttcttgctgctggtgggaggcagcgtggccatgggcatggtctgcacagccattgccctcttccagacactggccacgcaggtggggcaccgggctgaccgccgcaccttcaccgtgcccaccatcgtggtggaagacgcacagggcaagcgtcgctcctccattgatggctcagagcctgccagaacttcgctacagatcacgggccttgtggccaccattgtggtcatctatgactgcctcatgggtttccctgtgctggtgggtgaggtttgatgggtagggaacctctcagggatgtggcttctaggacagcctggggactgatggctgaaatgataaacctgacttttgagctgtcagttagcactcccattttatggatttggcagacagggttcagagaggttcaaagtcagggtagaagggtgagatgaacctcgacagctaggacctttttgtcacactgtaggcggtctttcccaggatgcagtaggtagatagggaacactgagttggcatagggcaggcccctaaCTTTCTGTGATATTGCGCTTTACCCTAAGGACCGGAGAAGGTCAAATTAAAGACTCACTGGAAGAAGGCCTTGCTAGCCACAG
1029 bp deletion beginning at Chromosome 4 positive strand position 152,279,522 bp and ending after 152,280,550 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37233 | TCC GAT GGC CAG TCA ACT AC | Mutant Forward | A | |||
37234 | TGA CCT TCT CCG GTC CTT AG | Common | A | |||
37235 | TCG ACA GCT AGG ACC TTT TTG | Wild type Forward | A | |||
37236 | Fluorophore-1 | CGC AGG ACA GCC TGG T | Quencher-1 | MUT Probe | ||
37237 | Fluorophore-2 | TTG GCA TAG GGC AGG C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37233 | 0.40 uM |
37234 | 0.40 uM |
37235 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
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 |