Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 130 bp
Wild Type = 77 bp
chr4:134836179-134836255 77bp GGTTCCTTTCCCCAATATTATC CAAACAAAACCCCACAAATC
Wt Sequence (deletions in lower case):
TTATTGGTAATGAATAGTGGTGTCCAGAGGAAGGCAGCATTATTAATATTACCACCAGCATATTTCTTGCCAATATTTTGGTTCATCTGCATTCCAGCAAGCTTTAAGAATGAAGCTCTTTAGGAATGGTTTATGAGGAAACAGGAAGCTTTTCAGTGTTAAAGTTCTTGTTCATTCAGGTAAAACCTGCCTATAGAATCCCAATGAATTCACTGTGGTCCAACCTCAGTTTCGGCCCAGTTCATGTCCTTGGCTTAAGAACCATGAACAAGAGCACTCTTTAATGTTGAATTAGCTACTGCTTTTTAGTATATGTGCTGCTCAAGCAAGCTTATAACCActgcttccccctggtctattctgagtgtttgaaatttcctcttgttaaattgctttgtattcagcaattttgttcaacaaagcttttaatagcatacttgaaattaacaaattctgaaatgtggcttttaatttggttttttttttctgtctttaggccttctcagtattttttgtttgtgtagcattcacttcaaatatcatatgtctcctcttcattcccatacaatggctttttttcgctgctagcacatatgtatgggtccagtacgtatggcacacaggtgagtcttaatgacttcttggttaggaattttagattcaagttaattatttagattgatattttaaggttcctttccccaatattatcttaatatgttgcttggcttgtggttgtggtttgttgatttgtggggttttgtttgttctttggttgtaggattggttatttgaggcagggtcttactagtaatcctggttgtcctgtaactcactatgtaggcCTGGCCGGTGTGGAACTCAGAGTGATCCTCCTGCCTCTGCCTTCTGAGTTCTGGAATTACAGCTGTGCCTTGCCTTACCCTGCTCTCGCTTGCATGCATGGTTTCCTTCTTTTTGATCTGAATGAAAAGCTTAGGTGTTAGGTCTTCTGGTTGGTCTCTAGTAAAGATGCCAGGTTAAAGGGTCAGGGCGGCTTCTGTTTAGGAGGCCTTGAAGACATTCGTGTGCCACACAAGACTGTTTACAACATTTTATTATTGAAAGGAATGCTAGTTAG
This mutation is a 505 bp deletion beginning at Chromosome 4 position 134,563,412 bp and ending after 134,563,916 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59011 | GGT TCC TTT CCC CAA TAT TAT C | Wild type Forward | A | |||
59012 | CAA ACA AAA CCC CAC AAA TC | Wild type Reverse | A | |||
59013 | Fluorophore-1 | ATG TTG CTT GGC TTG TGG TTG TG | Quencher-1 | WT Probe | ||
59014 | TGG CTT AAG AAC CAT GAA CAA G | Mutant Forward | A | |||
59015 | GCA GAG GCA GGA GGA TCA C | Mutant Reverse | A | |||
59016 | Fluorophore-2 | AGC TTA TAA CCA CTG GCC GGT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59011 | 0.40 uM |
59012 | 0.40 uM |
59014 | 0.40 uM |
59015 | 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 |