Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:41703683-41703822 140bp TAATTTCCATCAAGGAGAAGGAG TCAGTCACGAGGTTGGAGAC
Mut= 148 bp
Wt= 140 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AATTGAGATCTTAATTATATTGTTGCTTTAGTTCCCATTTACAATTCTCAGAGTCTAATTTCCATCAAGGAGAAGGAGCTTGAGAATCAGAATGCCTAATGAAGTCTGATTAGATTAGGCTACTGTTTCCTCTGctggttattgcaagtgcagtcagtattcctggggagggtgagtctccaacctcgtgactgattagagctgcaaaaagttgtttgttgagttcattaaagtatacatgttagtattacttaataagtttagaggtgttcttatgtttacattttttaaacaaagaaattaattttgctgttttgtgcatccattttttttttttttttagacctccttagctacgtgttttgctcaagagacatttgggaagcagtacaagcagacgataggactggatttctttttgagaaggattaccttaccaggtgagagactataaacctatgagactgtgtaggtctcgctcacagggcagccttactaggtaacagactatagaaccttacctgtgTGAGGTTATATAAGTCTCACAGGAATATTACCTGAAATTATCTATCATTAGCAACTAGTGTGAGCGTATACTGTCCACTGGGGTAAGAAAATGTTTATACAGTTCCCTATTCTATTGCTGTTAATAGCAACTCTGCAC
This mutation is a 392 bp deletion beginning at Chromosome 5 position 41,703,352 bp and ending after 41,703,743 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42311 | TAA TTT CCA TCA AGG AGA AGG AG | Common | A | |||
42312 | TCA GTC ACG AGG TTG GAG AC | Wild type Reverse | A | |||
42313 | ATA CGC TCA CAC TAG TTG CTA ATG A | Mutant Reverse | A | |||
42314 | Fluorophore-1 | TCA GTA TTC CTG GGG AGG GT | Quencher-1 | WT Probe | ||
42315 | Fluorophore-2 | CCT CTG TGA GGT TAT ATA AGT CTC ACA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42311 | 0.40 uM |
42312 | 0.40 uM |
42313 | 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 |