Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:56582929-56583016 88bp TGAGCAAGAGGGCTGTAAAGA AGCAGAGCAGACACCCATTC
Mut= 90 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TAAAGGTTTGCTTTGTTATGGGAGGCCTTTTGAGCGCCCCATTCAGCTCTTTGCCCTGCCTGAATCAAGGACAGGTTTCCTACCATCATCCCTGTGTTTGATGAAAGAGCACACAAGATGAGCGGTCACCAACCCTGATTTGGGGTTAATACTTGGATTTTATTCTATTTTTTTGTCTTGGGTTGGGGATTACATCTAGGGCTACAAGCTGCCAGGTCATGTATGACCACAACCTGctgacagtgactctgcaacccaagctttcagcttccctttctctaggttttgggtcttgagcagggaaggggagctggctgctaaatgtggtcttaagctagattgtgaacacttgcaactgcaaagttgcttcttaaagaacactttgtgtcaacccctcctaaaagataactttatttaaaacttcatactctttcccatcaggcatttaaggaggaaggacaagaacctgaggaggttggaatcagctggggagctgtgagcaagagggctgtaaagagaaacaccaaaggtacttaactgcctgtgcttgacgCTGGGCCTGGGGAATGGGTGTCTGCTCTGCTCTGTGTATGTGTAATATGCATACTTAGATGTGCATGTGTGTGGGACAGCCATAGTGTCACATTGTGTCCACCTTAGGTTTTGTTTTTGTCTTAAGGTTTATTTTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTGCGCGCTTGCCTGCATGTGAATGCACATGTGTTTGGTGCCCACAGAGGTCAGAAGCAGGTCTTGGACCCCTT
This mutation is a 317 bp deletion beginning at Chromosome 17 position 56,582,960 bp and ending after 56,583,276 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40784 | TGA GCA AGA GGG CTG TAA AGA | Wild type Forward | A | |||
40785 | AGC AGA GCA GAC ACC CAT TC | Common | A | |||
40786 | TTG GGT TGG GGA TTA CAT CT | Mutant Forward | A | |||
40787 | Fluorophore-1 | CAA AGG TAC TTA ACT GCC TGT GC | Quencher-1 | WT Probe | ||
40788 | Fluorophore-2 | ACC ACA ACC TGC TGG GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40784 | 0.40 uM |
40785 | 0.40 uM |
40786 | 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 |