Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:108070997-108071082 86bp TCATCCTGATGGAGGTAGCC TCAGGAATATGTCAGGGGTGT
Mut= 85 bp
Wt= 86 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case and insertion with carrots ^g^ (TCACCCTTCTCCTTGAACACTGGAGGATGCTTGGCTGAAGCTCCAGA insertion):
GATGATTGTGAGCCACCATGTGGTTGCTGGGAATTGAACTCAGGACCTCTGGAAGAGCAGTCAGTGCTATTAACGACCAAACCATCTCTCCAGCCCCAGAAATCACTTTTTCACTGAGGAAGGTTGAGACCTG^ctggtctggagcttcagccaagcatcctccagtgttcaaggagaagggtgacagggacttactgtttggctgagctcaagagcttgtgggtccccagcatctctcattccctaggaaaccgctaaacttccataacctgcctgagcacgtggaccagctgctacaggtggacagtgaagacaacgagagccagggtaggaggtgttctcaggggtggggtgggtgaggaatgtggtgtcaaagcctggagttctctgcacagctcatgtctccctgtgtctgtcagaccctctgccatccctgatttcacacatctcccttcccaggacaagttgaaggtcgacttggcccatctactgtggtcctagaccacacaggaggctttgaggggcttctccttgtggatgatgacctcctgggggtgagtggagatgtgtcttgggccccagctcagccccatcctctacgcttatcctgatggctacatgttcatcctgatggaggtagccatacacctatcttgacaccactccaag^CCATGGTGTCAGCCCTTAACACCCCTGACATATTCCTGACACCATGATACAGTCACTACCTCCAACCCTTTCAGCAAAGCCCAGAGTCCCATCATGTCTGTCCCAGAAGCTCTGGACATGGATACAGTTTCTCCTGCCTCAACCAGACACAGTGCAGTGTTGGCCATGACAG
This mutation is a 536 bp deletion beginning at Chromosome 9 position 108,071,036 bp and ending after 108,071,571 bp (GRCm38/mm10). In addition, there is a 47 bp inverted sequence from the deleted region Chr9:108071521-108071567 (TCACCC through TCCAGA) at the deletion site that is not predicted to alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41234 | TCA TCC TGA TGG AGG TAG CC | Wild type Forward | A | |||
41235 | TCA GGA ATA TGT CAG GGG TGT | Common | A | |||
41236 | CAC CCT TCT CCT TGA ACA CTG | Mutant Forward | A | |||
41237 | Fluorophore-1 | ACA CCT ATC TTG ACA CCA CTC CA | Quencher-1 | WT Probe | ||
41238 | Fluorophore-2 | AAG CTC CAG ACC ATG GTG TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41234 | 0.40 uM |
41235 | 0.40 uM |
41236 | 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 |