Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:76620341-76620465 125bp AAATACTTCTCAGCAACAGGAGTC GCACAGAGAGTTAGAGGTGACAGA
Mut = 114 bp
Wt = 125 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGCCCTTGTCATAAATAAGAGTGTTCATTACTTCCTGGGAATCTGTAACTGAGCTTTTTAAAATCTCATGTCATGTTATGCATTTCTACAAGAACTGACTCAACTTTCAATCTCCCtcatttctgaagtgtggtgggagccagttataaagaatccgtgctgtcctttgagtaactgagaatccatggctccagtcccacagctccttgaagggttagagacatagctcagtggtcgagcatttgcctaccacacacaaggtcgccaagaccatgaaaaatggaagtttgacattgtgtggtcctgatctgaaatttgcctataaataaatcccttctttgccttcctcctcctattcctccagggtatcaaccttgtgacccccaggtcatccaggaccgtgtcagccacctagagcagtgcttcagtgaactgagcaacatggcagctggacggaaggctcagctggagcagtctaagcgactctggaagttcttctgggagatggatgaggccgaaagctggatcaaggagaaggagcagatctactcctccctggactacggcaaggacctgaccagcgtgctcatcttacagcgcaagcacaaggcctttgaggatgagctccgtgggctggacgcccatctgaaacagatcttccaggaggctgacgacatggttgcacaaaagcagtttggacacccacagatcgagacccgtgtcaaagaagtgtcagctcagtgggaccacctgaaggagctggctgcctttcgtaagaaagacctgcaggatgcggaaaacttcttccagttccagggtgatgcagacgacctgaaggcttggctgcaagacgctcaccggctgctctctggtgaagatgtagggcaagatgaaggagccacgagggccctgggaaagaagcacaaggagttcctggaggagctggaggagagccgaggggtgatggagcacttggagcaccaagcccagggcttccccgaggagtttcgagattccccagatgtaacaaaccgactccaggcactcaggaagctctaccagcaggtactgacccaggcagaattgcgtggacacaagctgcaggaagccctggacttgtacaccgtgtttggggagtcagatgcctgtgagctgtggatgacggagaaggggaagtggctggaccagatggatatcccaaacacgctggaggatctggaggtggtgcagcacaggtcagacagctcagccttgctcatccccacccgagagctgagggtcacctcccactgctagaccctcctctgttacatagtgcaggcatcaggaattgagttctggtctgactccggggcctccgattcttcacccactggcacttcatagttctggagacaaaggggttttttaaaaaatatattattaaaatacttctcagcaacaggagtctttactcagatgaattctgagcattgggatacaggaAGAGAGGAGGTGCTGTGGACATGGGGCTGAGAGGCTGGGATTCTGTCACCTCTAACTCTCTGT
This mutation is a 1369 bp deletion beginning at Chromosome 12 position 76,620,406 bp and ending after 76,621,774 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50313 | GCA CAG AGA GTT AGA GGT GAC AGA | Common | A | |||
50314 | AAA TAC TTC TCA GCA ACA GGA GTC | Wild type Forward | A | |||
50316 | Fluorophore-1 | CTC AGA TGA ATT CTG AGC ATT GGG | Quencher-1 | WT Probe | ||
50317 | Fluorophore-2 | CAA CTT TCA ATC TCC CAC AGA GAG G | Quencher-2 | MUT Probe | ||
52319 | GTC ATG TTA TGC ATT TCT ACA AGA AC | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50313 | 0.40 uM |
50314 | 0.40 uM |
52319 | 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 |