Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:23226569+23226719 151bp AGCATGAGGTTTCCAGTAAAGA TGACAGAACCATCACCTTTGTC
Mut = 154 bp
Wt = 151 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CAATGTTCACTTAATACCAATTAGAGAAAAAAAAAAGAAATGAAATGGTTGCTATAGCATGAGGTTTCCAGTAAAGAATAAAACTAAAATTCTTCATCCcttgataatattcaggttacaatgccatcagaatatattcatactgagttcctctatgtgtattttctagctgcactccaactccgacaaaggtgatggttctgtcaagtacatcctgactggagaaggggccgggactatatttatcattgatgacaccacaggtgacatccactcaaccaaaagcctggacagagagcagaagactcactatgtgctccatgcccaggctattgacagacggacaaacaagcctcttgaacctgagtctgagttcatcatcaaagtgcaagatattaatgacaatgccccaaagttcacagatggaccatacatcgttactgtgcctgaaatgtcagatatgggtaaggacacttatttttatagtgaagaaatccattcccattatttgtatcaaatatatcttttaaagaattactttaatgtattttttcaaacattgagaaagctctaaaatgttataatatttttattgcagtaagaacatctattattgaaccatacattatatattgaaaaaattcttaatatggctgtgtagaaatacttgtaatatctcaagccaaagccataaCTAGTTATCATTTGCAATTCTTATTTATTTGCATTATTCTTGAATATGCAAAATTTTTCATGTTATTA
This mutation is a 595 bp deletion beginning at Chromosome 15 position 23,226,613 bp and ending after 23,227,207 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51092 | AGC ATG AGG TTT CCA GTA AAG A | Common | A | |||
51093 | TGA CAG AAC CAT CAC CTT TGT C | Wild type Reverse | A | |||
51094 | TTC AAG GCA GTT CCT GTT GA | Mutant Reverse | A | |||
51095 | Fluorophore-1 | CAG GTT ACA ATG CCA TCA GAA TAT ATT C | Quencher-1 | WT Probe | ||
51096 | Fluorophore-2 | CAT CCC TAG TTA TCA TTT GCA ATT CTT AT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
51092 | 0.50 uM |
51093 | 0.50 uM |
51094 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | repeat steps 2-3 for 40 cycles |