Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:64976962+64977076 115bp GAAGGCCAAGGTCACTTAAGAC GCTTTTTCACAGTATTATCCCTTTC
Mut= 106 bp
Wt= 115 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCAGGACATTAGTAATCACTAATGCTTGTCTGTTAGGTTGCTCATTGCCTAATGCTGAAATGACTTAAGTAGTTTCTGCTGATGAGTCAGACATCCTTTCCTTTTGTGTTTTTTCTCCACTCTTACTGCCAgataaaagaaaggtaaagaaaagacttgaagtctagtaaaagaaggaaaaaactagcatttctatcttggaaggaaaaatactgttaatgtgtcatgctgaatgagtgtttgcattacatactcaataattctgttctttatctgggttttgtaattggttttaaattatttgttaccaaggaggcttagcaaaaatttgctaacttactctcttctcagttttcagctcatgaccttatcccgtctccaaaattaaagccctctcaaggaatgccagccagtgatgatttatgttttagcaaaaaaaggtcatcaagaaacttatttcagagtagtcgggactttaactcagagtcagttcctacatgtggtgctggtgagtaaatatttgaaataaatgctattgaagactagctaaaaagcagacactttgacataattgtgtcatgagatcataggtcatctcattgtaggctgttagtaatggaaggccaaggtcacttaagacactaatatttcttgagcataagtctacatgtcgGATATATTTCTTAATGTTCTTAATGTAGCTTTCAAGAAAGGGATAATACTGTGAAAAAGCTTGTATTCTATGTTGGAGGTGGTGGTATACACCTTTAATGCTTAGGAGGAAGGAACAAACAGATCTCTATGAGTTCCAGGCCAGGTTG
This mutation is a 542 bp deletion beginning at Chromosome 12 position 64,976,475 bp and ending after 64,977,016 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43391 | GAA GGC CAA GGT CAC TTA AGA C | Wild type Forward | A | |||
43392 | GCT TTT TCA CAG TAT TAT CCC TTT C | Common | A | |||
43393 | GTC AGA CAT CCT TTC CTT TTG TG | Mutant Forward | A | |||
43394 | Fluorophore-1 | TTC TTG AGC ATA AGT CTA CAT GTC G | Quencher-1 | WT Probe | ||
43395 | Fluorophore-2 | CTC CAC TCT TAC TGC CAG ATA TAT TTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43391 | 0.40 uM |
43392 | 0.40 uM |
43393 | 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 |