Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:34769017+34769115 99bp CCTGGGAACTCTTCTGTCTAGAGT CTAAGGACTAACTTGACAGGGCAGA
Mut= 90 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTAGACCAGGCTAGCCTCGAACTCAGAGATCCACCTGCCTCTGCCTCCCAGGTGCCAGGATTTAGAGGCATGGCATTACCACTTCACAGCTCATACAGGTTTCTTGATTGACTGCATTTTTGAggaggagtggcagacagtgtataaacattcttggtgccaggtgggttctcaggctgcaggtagcatggtgtgggtgggtttagatcctgttctgttcccttaatagctgtgcctgttaaccttggacaagcacagtcatagtacttccttcatggagacttttgagaaattacattctgtggtacagtagctcttagaatatgttagccattaacccttttcattatgcatgcctttcagaaggtgcttcctggaggggcctgccccactgttcctgtgctgagttccaggacagcctcaacttcagctaccatccctcaggcctaagcctgcacctcagaccacccagtcggggaaactcccccaaggagccgcccctctcccaagtactgagccccgaacccccagacccagagaagcttcctgtgccccctgcccctccatccaagaggcactgccgctcactctcggtgcctgtggacctgtctcgctggcagccggtgtggcggcctgccccctccaagctgtggactcccatcaagcaccggggcaatgctggagggggtgggccgcaggtgcctcagcagagccctccgaagcgggtctccagcctcaggttcctccaagctcccagtgcctcgtctcagtgtgccccagcccacagaccctacagccctcctttcttcagcctggccctggcccaagattccgctcagccctgcgccacctcccctcagagtggctcctgggagagtgatgcagagtccctgtcgccctgcccaccccagcgccgcttctccctgtcacccagcctgggcccacaggcgagccgcttcctgccctctgcccggagctctcccgcgtcttccccagagctgccctggagaccccgaggtctccgaaaccttccccgaagccgctcacagccttgtgatctggatgcccgcaaaactggtgtcaagcggcgtcatgaggaagactgcaggcgccttcggccttccttggactttgacaagatgaatcaggtgggactggcagaccagaagctgtttaaacatctcctaattccctaccttctggcaaggcctccctgggaactcttctgtctagagtttatacaagaaaaccaccctggcagtattgaattttaaCCTTTAATGTCTTCTGCCCTGTCAAGTTAGTCCTTAGTATTCTAAAGAAACTAGACTGAGGCCTTAAGCCTTCAGACAGGGATCCTAATTGTCCTGCCAGAGGCACTTTTCCTCTTCTCTGAGAACTCTAGACCACAGG
This mutation is a 1151 bp deletion beginning at Chromosome 18 position 34,767,928 bp and ending after 34,769,078 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43728 | CCT GGG AAC TCT TCT GTC TAG AGT | Wild type Forward | A | |||
43729 | CTA AGG ACT AAC TTG ACA GGG CAG A | Common | A | |||
43730 | TGG CAT TAC CAC TTC ACA GC | Mutant Forward | A | |||
43731 | Fluorophore-1 | ACA AGA AAA CCA CCC TGG C | Quencher-1 | WT Probe | ||
43732 | Fluorophore-2 | CTT GAT TGA CTG CAT TTT TGA CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43728 | 0.40 uM |
43729 | 0.40 uM |
43730 | 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 |