Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:32470591+32470715 125bp CAGGAGCTCTATCCCTGAGC CTCTCTTCCCTCTAGAACACCA
Mut= 120 bp
Wt= 125 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCTTTGGAATCCAGGTTCTCACACATATGAGGCAATATCTCACACATTCTACCCCACAGGCATGTCCCCAAACCCTCACTGGGGGATTCTGGTCAGGAGCTCTATCCCTGAGCCACGCCCCAGCCCCATTGTTCTTTCTTGTTTTAGAGtggggagcaactcagggaagctcagcaaggcaccatgtctttgcttttggtgttctagagggaagagagagtagtgagagggagaggagggggagggagggagggacagactaagccgagctgtcaggtcctcattgcccctttctatctgccacagtatcttcccaatgagaagggccttcaaaacgagaagaaggtgctggacaccatgcaccacatcatcctggcatgggttggacctttcttgcctctcctggtgcttgttcatcctgattacatcaagcctgtgttgggtgcctcaggtaggcaggttggaccgcagagggatgagtcaacttgaaactcccagaacccttctagggttacagacatgtgggtgccccacaggaagtgtgccttgagtaagtgtttgagggatggatagaagttcaccaaacTGGGGCCGGCAAAGCAGGTTGTGAGAACATGGACAATTGTGGGGTGGTGTGCAAGAGTTTCAGGAACATTGTAAGTCTCAGGCGCACCTGCTGGAGTCACATCTAAGCAACTGACTTATTCAAGGTTTTGGTAAGTAATTGTCGTTCAATTGTAGGCCATGACAGTCCAT
This mutation is a 437 bp deletion beginning at Chromosome 17 position 32,470,647 bp and ending after 32,471,083 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43042 | CAG GAG CTC TAT CCC TGA GC | Common | A | |||
43043 | CTC TCT TCC CTC TAG AAC ACC A | Wild type Reverse | A | |||
43044 | CCT GAA ACT CTT GCA CAC CAC | Mutant Reverse | A | |||
43045 | Fluorophore-1 | AAC TCA GGG AAG CTC AGC AA | Quencher-1 | WT Probe | ||
43046 | Fluorophore-2 | TTA GAG TGG GGC CGG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43042 | 0.40 uM |
43043 | 0.40 uM |
43044 | 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 |