Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:8797183-8797275 93bp AGGTGAAGTGGTGGATGCTG GAACCCTCACCTTAGGAAATACC
Mut = 117 bp
Wt = 93 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTCTGACTCGTTTGTGGTTTTTGAAAGACCTGTCTCAGCTTTGACCCTGGCAAGTCCTCCTGCCTTAGCCTACATGGTACTGAGAGAATAGGTGTATGTCAGCATATCTAGGCCTTCTTGGGCATTTACTGTACCATCATGAGAGCATCATTTAATATTTCATGTGCTAAGCTGGTCGGGGGTGGCGCATGCCTTTAATTCCAGCACTTgggaggcagaggcaggtggatttctgagttcaaggccagcctggtctaaagagtgagttccaggacagccagcgctacacagagaaactctgtctcgaaaaaacatatatatgtgtgtgtgtgtgtgtgtgtgtgtgtatatatatatatatatatttaggtgctaatctgtgtcctgtctcttcttgatcaatgaaccatctgaacagaggttctttcacacagagactcttattgctggtggcttttcagaggctcttcacatagctgtcttctttcatttttcatgcagatatggctttgtaattgctgtcaccaccatcgacaatattggtgctggtgtgatccagccaggccgaggttttgttctttatccagtgaaatacaaagctattgttttccggccctttaaaggtgaagtggtggatgctgtggtcactcaggtcaacaaggtaagagtattattcatagaaaggttatGGGGTATTTCCTAAGGTGAGGGTTCAGTCCCTAGTCCACTTACTAGTTGCCTTAAGTCTTTTGTTGTTGTTAAGATTCATTTTATCTTAGTTTTTTGGTTTTTCTTTTCTGGTTTTAGGGTTTTCTTTTTTCTTTTCTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCTCTCCTGGAACTCACTCTGta
This mutation is a 480 bp deletion beginning at Chromosome 19 position 8,797,208 bp and ending after 8,797,687 bp (GRCm38/mm10). There is a single bp insertion A at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50762 | GAA CCC TCA CCT TAG GAA ATA CC | Common | A | |||
50763 | AGG TGA AGT GGT GGA TGC TG | Wild type Forward | A | |||
50764 | TGG GCA TTT ACT GTA CCA TCA | Mutant Forward | A | |||
50765 | Fluorophore-1 | TCA CTC AGG TCA ACA AGG TAA GAG T | Quencher-1 | WT Probe | ||
50766 | Fluorophore-2 | CAT GCC TTT AAT TCC AGC ACT TAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
50762 | 0.50 uM |
50763 | 0.50 uM |
50764 | 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 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |