Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:14348981+14349065 85bp CCATGACACCTGGGTTGG CAGAGGTCACACCTGGCATC
Mut= 85 bp
Wt= 85 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTTAGGCAAATTTATGAAGGCTAGAACATGATGTCATCTGGGTTTATGCAAATAAACTCAGATGGAACCATGATGTTATGCAAATCCATGCAGATTTATGCAAAGCCCATGATCAGGGCCTGTGGATATTTATTCAAATGAAATTCAAATTTATGCAAGTTCATACAAATTAATGCAAATTAGTGCTGGCCCTACCACTCACCCCAAgtatgggtgacccctgctgacgccgacatgcaggttgaccccaggtgaccccgcccctcacactgaccctgccctgccccctgacctctgtgaccccgcaggctgaccccgggcttgcccgctgctcattccagtcgctctccctctgccatgtgaccaacttcaccgtcttcctggggaaggacagggctgtggcaggatccatccagttccccccagatgatggtgggtgaggccccgcctccgtgacccccacgtgacccatgacacctgggttggcccccccaagaaccaactgtgacccctaaccctagctgtgaccctatgatgccaggtgtgacctctgatcccatgtgtgaccaggggtcctaccctggccccagcccatgacccttgaccccagggtgcgacccctgctcccaaATCTTACACCTGACCCCAACATGGGACCCCAACCCCGTGTGTGACCCCTGACCCTGTCCCCTCCATCATGACCGCAGTGACCCCATCCCAACCCCCACAGATGGCGACCACGAGGCCGCAGCCCAGGACCTGCGCTGCTGGGTGCATGAGGGGCAGCTGAGCTGCCAATGGGAGCGGGGTCCGAAAGCAACGGGTGACGTGCACTACC
This mutation is a 423 bp deletion beginning at Chromosome 14 position 14,348,720 bp and ending after 14,349,142 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52639 | CCA TGA CAC CTG GGT TGG | Wild type Forward | A | |||
52640 | CAG AGG TCA CAC CTG GCA TC | Wild type Reverse | A | |||
52641 | AAT TAG TGC TGG CCC TAC CA | Mutant Forward | A | |||
52642 | AGG GTC AGG GGT CAC ACA | Mutant Reverse | A | |||
52643 | Fluorophore-1 | AAC TGT GAC CCC TAA CCC TAG C | Quencher-1 | WT Probe | ||
52644 | Fluorophore-2 | CAC CCC AAA TCT TAC ACC TGA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52639 | 0.40 uM |
52640 | 0.40 uM |
52641 | 0.40 uM |
52642 | 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 |