Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:56925264-56925360 97bp ACCAGGGCCTACTCTCCAAC TCCAGGCTCTCCCATATTCC
Mut= 97 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTTTTTTTTTGGACACCCTGTTGAACATCCTGAATCTGGGCAAGGCATTTTCCTTCCAGTCTTGACCTAACAGATAGCTGAGCCATGGCACTGAAAGGCCGAGCCCTCTATGATTTCCACAGTGAGAACAAGGAGGAAATCAGTAtccagcaggacgaagagctggttatcttcagtgagacctcactggacggctggctgcagggacagaacagccgtggggagacagggctcttccctgcttcttacgtcgagattgtccgtcctggcatcagcaccaaccatgtggactattccaacagccctgcaggctccctgggcacccaggggagtttgtatagcagcccgagtatggccagtccagccaggagtggtgggggcagcggcttcctctcaaacccaggaagcttcgaggatgatgatgatgatgattgggatgactgggatgatgggtgcacagtggtagaagagccactagctggcggcctaggtaccaatgggcaccctccactcaacctctcctacccgggtgcctaccccaaccagcatatggctttccgacccaaggcacccctggaaaggcaggacagcctggcgtctgccaagcggggcagtgtggtaggacggaacctcaatcgtttctcgtgcttcgtacgctctggagtggaggccttcatccttggtgatgtgcccatgatggccaagattgctgagacttactccattgagatgggcccacgtggccctcagtggaaggccaacccccacccatttgcctgctccatagaggaccccaccaaacagaccaagttcaaaggcattaaaagttacatctcttacaagcttacacccacccacgcaggctcgcctgtttaccggcgctacaaacactttgattggctgtacaaccgccttctacacaagttcacagtgatctcagtgccccacctgcctgagaagcaggccacaggccgcttcgaagaggacttcatcgagaagcgcaagcgaaggctcatcctctggatggaccacatgaccagccaccctgtgctctcccagtatgagggcttccagcacttcctcagctgcctggatgacaagcagtggaagatgggtaagcgccgggcagagaaggatgagatggtgggcgccagttttctgctcactttccagatccccacagagcaccaggatctgcaggatgtagaggaccgcgtggatacattcaaggctttcagtaagaagatggatgacagcgtcctacagcttagcaacgtggcggcggagctggtgcggaagcatgtggggggcttccgcaaggaattccagaagctaggaagtgccttccaggccattagccatgccttccagatggaccctccctttaggtctgatgctctcaacaatgccatttctcacactggccggacctatgaaaccgttggcgaaatgtttgcggaacagcccaagcacgacctcttccaaatgcttgacacactgtctctctaccagggcctactctccaacttccctgacattatccacctgcagaaAGGTAAGCCTGCATGAGTGTCCTGGTCCCTGGGAATATGGGAGAGCCTGGACTATGAACAGCTCATCACACTTTCCTGTCTCCATCTCCTTCGCTGCTCAACAAGTGTGTTAAGACAGTTCTCTGCAGAGTGCTGTTGT
This mutation is a 1408 bp deletion beginning at Chromosome 9 position 56,925,315 bp and ending after 56,926,722 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42671 | ACC AGG GCC TAC TCT CCA AC | Wild type Forward | A | |||
42672 | TCC AGG CTC TCC CAT ATT CC | Common | A | |||
42673 | CGA GCC CTC TAT GAT TTC CA | Mutant Forward | A | |||
42674 | Fluorophore-1 | CCC TGA CAT TAT CCA CCT GC | Quencher-1 | WT Probe | ||
42675 | Fluorophore-2 | AGG AGG AAA TCA GTA AGG TAA GCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42671 | 0.40 uM |
42672 | 0.40 uM |
42673 | 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 |