Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 115 bp
Wild Type = 137 bp
>chr9:44715014-44715150 137bp GAGAAACCGCCTTTGAAATC AAACGAAAACACCGAAGAGC
Wt Sequence (deleted region in lower case):
TTGCTCTTCCTAAAAGAGAGGCACAGAAAGTTGGGAAAACCAAGGCAAGGAGGACCATCTTGCTGGAGCGAGTTAGAAATGACTAGACCTAGAAGTACGTACCTGGTACTGTTGCTGACTCTGTCCTGAGTCATTCAGAGTTAGAGCCAGGGAGGCCTTGCCTACCATTTCCCTTTATAGAAGGGGAACCCAGGGCAAGAGAGTTTGAATAAAgctgtagaacaggtaggtcagtggcagagtgggttgaactcagctctcctggctccttctccagagccctcgaggccccgttgccctaggatgaggtaggatggtactttcttgcttgtgcatgtatatggctccctggagccaaaaactggagcttgaggcctctttgcgtttctccctcagctgaatcggagagcctggtcaatgggaaccacacagcacaacctgcaacccgggcaccgtcagcctgtgccagccacagttccctggtgagctctattgaaaaggacctgcaggaaatcatggattcattggtgctagaggagcctggagctgcaggcaagaagcctgctgccacatccccattgtcgccaatggctaacggcgggcgctacctgttgtcccctccaaccagccccggtgccatgtctgtgggctccagctatgagaacacctctccagccttctctccactttcctcaccagccagcagcggaagctgtgctagccattcaccaagtgggcaggagccaggaccctctgtgcccccattggtgcctgcccgctcctctagctaccatttggctctgcaacctccacagtctcgcccaagtggttcccgctcctctgacagtccccggctgggcagaaaagggggtcatgagagacctcccagccctggtctccggggtcttctgacggacagtcctgccgctaccgtcttggctgaggcccgcagaaccactgagagtccccggctaggtgggcagttacctgtggtggctatcagcctgagtgaatacccatcttccggtgcccgcagccaacccgccagtattcctggcagccccaagttccagtctccagtgcctgctccccgaaacaagatcggcacactccaggaccgccctcccagtcctttccgtgagcccccaggcacagagcgggtactaacaagcagcccctctcgacaactggtgggcagaacattttcggatgggttagcagcaactcgtaccctccagccgcctgagagtccccgcctgggccggaggggcctggacagtatgcgggaactgcccccgttgagtccatctctgtctcgaagggctctgtccccactgccagcccggactgccccagatcccaagctaagcagggaagtggccgagagtccccggccccggcgctgggcagcccacgggacttcacctgaggacttctccctgactctaggagcacggggtcgcaggactcggagtccctcgcccaccctcggggagtccctggcacctcgcaagggtagcttcagtggcaggctgagcccagcttacagtctgggctctcttactggggcttcacctcgtcagagcccccgtgctcagcggaagctctccagtggggacttgcgggtacccattccaagggagaggaaaaacagcatcacggagatcagcgacaatgaggacgaacttctggagtaccaccgtcggcagcgccaagagcggctccgggagcaggagatggagaggctggtgagccaatgccagggaggctgccgctgcccaaggcggtgcctctgccagggtgtgggcaggccagccggcagaacaggaggggccatgtacagggcctgggcggattcaggatgaaccatcaggctaagtgacctgtgtctcaccatcctgataccctggagagaaaccgcctttgaaatcactaaggctttctaaattttttagttttgtatacgtgtgtgcaggtgtgtgtctgtgtgtgtagGCCAGAGATGAGTGGGTAGTGTCTTCCTCAATTGCTCTTCGGTGTTTTCGTTTGTTTGTTTTGTTTTTTTGGGGGGGGGGAGCGTTAAGACAGGGTTTCTCTGTGTAGCCTTGGCTGTCCTGGAACTCATTCTGTAGACCAGGCAGGCTGGC
1782 bp deletion beginning at Chromosome 9 negative strand position 44,716,848 bp and ending after 44,715,067 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37251 | GAG GCC TTG CCT ACC ATT TC | Mutant Forward | A | |||
37252 | AAA CGA AAA CAC CGA AGA GC | Common | A | |||
37253 | GAG AAA CCG CCT TTG AAA TC | Wild type Forward | A | |||
37254 | Fluorophore-1 | CCA GGG CAA GAG AGT TTG AA | Quencher-1 | MUT Probe | ||
37255 | Fluorophore-2 | TTT GTA TAC GTG TGT GCA GGT GT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37251 | 0.40 uM |
37252 | 0.40 uM |
37253 | 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 |