Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:122432484+122432599 116bp GAACATTTCTCAATGTCCCAGA CCGGCTCCTTAAAACAAAGT
Mut = 110 bp
Wt = 116 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGAACATTTCTCAATGTCCCAGAGGCAGCATCTTTGGGTCAGATAttaggaggtgttttgcgaggatcacatggctaagaaaaacaaggataatagtactttgttttaaggagccggtctttatttgcacagggctgtgggttggtttttctgctagaaagtagaatagtagtcgggcatcaaatgagcgcgcgctcacggcgcagtgtttgctgtgcggatactcttgctttaccgttctctgtttcgtgtgtgtagataaacatgatgctggcaaacctgtacaagaaggctggtcaggagcgcccatcagtcacaagctataaggaggtcctgcggcagtgccctttggcccttgacgccattctaggtacagaaagttttctccctggtgcctggggtgtttgcagtcttagatgatggttctcagtcacaactatggttagatagagcagggtttttttttcagattTTAATTTTATGTTAATGTGAATGAGGGTTTTGTCTGTATGTCTGTATGTATGTAGTGTCTAATGCCATAATGGTTGGAGGGGGGCATTGGAGCCCCAAGAG
This mutation is a 427 bp deletion beginning at Chromosome 5 position 122,432,528 bp and ending after 122,432,954 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51530 | GAA CAT TTC TCA ATG TCC CAG A | Common | A | |||
51531 | CCG GCT CCT TAA AAC AAA GT | Wild type Reverse | A | |||
51532 | GGC ATT AGA CAC TAC ATA CAT ACA GAC | Mutant Reverse | A | |||
51533 | Fluorophore-1 | CGA GGA TCA CAT GGC TAA GAA A | Quencher-1 | WT Probe | ||
53370 | Fluorophore-2 | AAT GTG AAT GAG GGT TTT GTC TGT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
51530 | 0.50 uM |
51531 | 0.50 uM |
51532 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
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 |