Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:24077389+24077519 131bp GTTGCTGGGGTACCTATTATAACTC ATTTGCAGTTGATGAATGTCC
Mut = 169 bp
Wt = 131 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TTCATTTTTTATAATTAAAATACAAAAAGGATTTAtaaccgtgggttatatttaaaattaattcttcagtatggagttcatttctttaatttttatatctcaaaattactgttgtcttctagcaggacagaaatcccccaagcatcaaaaagcagattcatgaccagcaacttttcccaaccagccctgcagctctgctatgagaacacgaatggatcctgtattaaaactccctattctccagggccccgggtcatcctgtacatggtctatggctttggggctgtgctggcagtgtgtgggaacctcctggtggtgatttcagttctccatttcaagcagctgcactctccagccaattttctcatcgcctctctggccagtgctgacttcttggtgggcatctctgtgatgcccttcagcatggtcaggtccattgagagctgctggtactttggagatgcattttgtagccttcacagttgttgtgatgtggctttttgttactcttctgtcctccacctctgcttcatctcagtggacaggtacatcgctgtcacggaccctctggtctatcccaccaagttcacagtgtctgtgtctggaatttgcatcagcatctcctggattctgcccctggtgtacagcagtgcagtgttttacacaggcatcagtgctaaggggattgaaagcttagtaagtgctctaaattgtgtagggggctgccaaattgttatcaatcaagactttgttttgataagttttcttctattcttcatacctacccttgttatgatcattctttacagcaaaatatttttggtagccaaacagcaagctgtaaaaattgaaacttctgtaagtggcaacagaggtgaatcatcctcagagagtcacaaagccagagtggccaagagagagaggaaggctgcaaaaaccttgggggtcactgtggtggcttttatggtctcgtggctcccatacacaattgacgcattggttgatgctttcatgggcttcatcactcctgcctatgtctatgaaatctgttgctggggtacctattataactcagccatgaaccctttgatttatgctttcttttttccttggtttaagaaagccataaagcttattttaagtggggaaattctaaagggacattcatcaactgcaaatttattttcagagtaagcattaatacaaaattcagaaaattccttaaggtataaaattatgaaatggtgacagaactaaatctaataaatgaccttTATGAAGCTAGAAAAATACAGCATAAAAACATTAGTCAGGAGGA
This mutation is a 1239 bp deletion beginning at Chromosome 10 position 24,076,376 bp and ending after 24,077,614 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51999 | GTT GCT GGG GTA CCT ATT ATA ACT C | Wild type Forward | A | |||
52000 | ATT TGC AGT TGA TGA ATG TCC | Wild type Reverse | A | |||
52001 | CAG TGA CGT TTA ACA TAG TTA TAG GAA | Mutant Forward | A | |||
52002 | TTG ACA AGA CAT TCC TCC TGA | Mutant Reverse | A | |||
52003 | Fluorophore-1 | CCT TTG ATT TAT GCT TTC TTT TTT CC | Quencher-1 | WT Probe | ||
52004 | Fluorophore-2 | ATT GTG TGA CCA TCA ATG GGA AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51999 | 0.40 uM |
52000 | 0.40 uM |
52001 | 0.40 uM |
52002 | 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 |