Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:67329447+67329555 109bp TCAGATGACTTGGGGGTGA ATGAGCATGGGTTTAGAATGC
Mut= 98 bp
Wt= 109 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTTAAACCAAGACAACGTCCCTTGACTTTAGCTATCCTAAGATCTATCTGGCAATTCTGAAGGCCAACTACTCTTTAGCTTTACATACCAGATATTCCTTCTGGGCACACACAATGCCAGGCTGGTCTGGGAGGCACCAGACTAGAATcaggttaaccaaggggagttattgctaggtcatctccaggcacagaaatgatccattaagttgtggcaacgccctcactctgctgcagtatctcactatgccgcgcctaccccaggagagaacaggtcagtgacgctgcaccctcgccttgcttttcctcgttctacccaggagctcactctgaacagtgaccaagtcttccccatgaacccccctaagtttgacaagatcgaggacatggccatgatgacccacctgcacgagcccgctgtgctgtacaatctcaaagagcgttatgcagcctggatgatctatgtgagagccttttgacctctttctattttgctgcctctctgaggaaaaaacaagatccttaatcttttgaatttcccacagacctactcaggcctcttctgtgtcaccgtcaacccctacaagtggctgccggtgtacaaccctgaggtggtggcggcctaccgaggcaaaaagcgccaggaggccccaccccacatcttctccatctctgacaatgcctaccagttcatgctaacaggtaagtgaagcctttgatttgtatgattgtaaggcattattggcctagtgtttgggcccttatataccccacacatctaaacaatacagtagcagccagcatgaacttgggagcctcagatgacttgggggtgaatattccaagccatggcacttttattatagtagtcatggtgaagggcaaagCCAGGGGTTCTTTCTGAAGCATTCTAAACCCATGCTCATTTCTGGGGGTGGGGGTGCATTGTAGAACACAAGGACACTGCTCTTCTATATTCCCAACTTACCCTGCTATGGGTACATTTAGACGAGACTCCCCAAA
This mutation is a 738 bp deletion beginning at Chromosome 11 position 67,328,779 bp and ending after 67,329,516 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44572 | TCA GAT GAC TTG GGG GTG A | Wild type Forward | A | |||
44573 | ATG AGC ATG GGT TTA GAA TGC | Common | A | |||
44574 | AGA TAT TCC TTC TGG GCA CAC | Mutant Forward | A | |||
44575 | Fluorophore-1 | AGT AGT CAT GGT GAA GGG CAA A | Quencher-1 | WT Probe | ||
44576 | Fluorophore-2 | CTG GTC TGG GAG GCA CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44572 | 0.40 uM |
44573 | 0.40 uM |
44574 | 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 |