Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:80576732-80576828 97bp AGCCACCGCTGTCCCTTC ATACGAGGGGCTTACCTGTG
Mut= 80 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case) point mutation in brackets with wt first:
CGGGGCGGGACTGAGCGCGGGCGGCGGCGGCGCGCCGGGGCCTCCTCCTCCCTTCCCTCCTCCGCCTCCCGCACTCTAGCAGCCGCTGCCGGCCGGACGGGCGTAGGTCCCACCGCCTCCGCTTCTCTCCCGCCGCCGATTCCGTGTCTCCG(C/A)GTCCTTGcccggcatgtcggcggccgtggcgtgtgtggattactttgccgccgacgtgctcatggccatctcctcgggagccgtggtgcatcgcggacgacccggccccgagggcgcgggccccgctgccggtctggatgtgcgcgccacgcgccgcgaggccacgccgccggggacccctggggcaccgccaccgccagctacggctccggggccgggaggcgccaccgctgcgccccacctgctggccgccagcatcctggccgatctgagaggcgggccggtagtcgccaccgcagccagcaccgcggggggcacctctccggtctcctcctcctcagccgcttcgtctccctcttcgggccgcgcccccggcgccgccaaaagccaccgctgtcccttccatggctgcgccaaagcctattacaagtcttcgcacctcaagtcacacCTGCGGACTCACACAGGTAAGCCCCTCGTATTTTACACAGGTGGGATGCAGGGAGTGCATTAGAGAGGTCTCGGGGACCCCGTTCCCCCATCTCACTTGGAACCCCGAAACTGCGTGGCGCGTGGGGGGCGGGGAGGGGGGCGCGCCCAGGC
This mutation is a 444 bp deletion beginning at Chromosome 10 position 80,576,763 bp and ending after 80,577,206 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42662 | AGC CAC CGC TGT CCC TTC | Wild type Forward | A | |||
42663 | ATA CGA GGG GCT TAC CTG TG | Common | A | |||
42664 | ACC GCC TCC GCT TCT CTC | Mutant Forward | A | |||
43625 | Fluorophore-1 | CGC CAA AGC CTA TTA CAA | Quencher-1 | WT Probe | ||
46159 | Fluorophore-2 | CCG AGT CCT TGC TGC GGA CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
42662 | 0.50 uM |
42663 | 0.50 uM |
42664 | 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 |