Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:63030174-63030281 108bp AAAAGAGGTATCTGTTGCATGG CTTTTGATAGCACCGAACCA
Mut= 96 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGATGTGTTCTAGAAGGTTCTAACAAATTGAGAGGTCATTGTCTCATCCTTACTCTATCTGCCTCACTCTACCTGCCTTGGCTTAGTTACTCATACAAACTCTTTTCACCCGCAAACGTCTCCACTCTCCTAGGACACTCACAATTCTGTACAAGCTTCCTTAGACATAAATCCCTTGgctttccaacgtcaagggattgaatgttggaagggacactgtaagaaagacagccagttggcccacccaagatgctagctagcctcagctgtcatcttctcttcttctaacagtgtcatggtctatgggacgaggatgacattgttgacagtaacacagacaaggaaggatcggatgacgagctgtccctcgatcagggcagaagaggctcctcagactcactgaagtctatcaacctggctgcctctgtagagtctgtccacgtgaccttccccgagcagccagcggccatcaggagaaaacgctcctgcagtagctctcagatctccccacggtccagcttctcttcaaataggtccaaaagaggtatctgttgcatggccattaggttgaaatgagccccacatagtctcattgATCTGGTGCCTCTGCATCTGGGGAGGGAAGTGGTTCGGTGCTATCAAAAGACGATGTTGTGACATGGAAGAAACATGCTCTGGCATATGACAGACCCAAGTTCAGAACCTGTTTCATCTGTATGTAGCTTCATTGTTCAATATGTGTGAGTCCATTCAGGGACCAGGATTTGGCCAGCACATAACCTCATAACCTA
This mutation is a 417 bp deletion beginning at Chromosome 18 position 63,030,224 bp and ending after 63,030,640 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46902 | AAA AGA GGT ATC TGT TGC ATG G | Wild type Forward | A | |||
46903 | CTT TTG ATA GCA CCG AAC CA | Common | A | |||
46904 | GGA CAC TCA CAA TTC TGT ACA AGC | Mutant Forward | A | |||
46905 | Fluorophore-1 | AAA TGA GCC CCA CAT AGT CTC A | Quencher-1 | WT Probe | ||
46906 | Fluorophore-2 | AAT CCC TTG ATC TGG TGC CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46902 | 0.40 uM |
46903 | 0.40 uM |
46904 | 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 |