Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:143988068-143988162 95bp AAAAAGGAGAGCCTGGTGAG CAAACAGCAGAGCACTAAGGA
Mut= 112 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (TTAGTGCTTAG insertion):
TGCTTAGCTTTGCTGTCTTTGTCATGTTGCCACTACTCTCTGATGTTTCACTTCCGGACAGCACTTGTGCTCATGAGTGCTTTAAATGGGGCGGGTTCAAGGGGGCACCTTGAACTTGGGCCATGGCCATGGGCTTTGGAGGCTACAGAACATTAAAAACTTGAGTCGTTCCC^ctgtacttctccatccgcctgtttgtatctccagtactaaaagaactattgataccgtaggtaaaaattacccatttctgaagagagggtaagaaagaggagcaaacatggctttgctcctactctgaatttagtctgaagacagtgagaaaccttgagtttggtgataggggatggaaactgtccagtccctcagtttccccagaagtaatctacttgtctgtgttggcaggtggaaagcaagccagccaagatggatatctacgacactcagaccttgggggttgtggtctttggcgggtttatggttgtttctgccatcggcatcttcctggtgtcaaccttctccatgaaggagacgtcgtatgaagaagcccttgccaaccagcgcaaggagatggcgaaaactcaccaccagaaaggagagaagaaaaagaaggagaaaaccgtggagaagaaaggaaagaccaagaaaaaggaagagaaacctaatgggaagatacctgaacatgacctggatcccaacgtgaccatcatcctcaaggaaccagtgcgtgtatccgctgtggctgtggctccaacttcagtccattcttctgtgggccatacccctatagccacagtaccagccatgcctcaggaaaagctggcttcctcccctaaggacagaaagaagaaggagaaaaaagtggcaaaggtggagccagcagtcagttctattgtgaattccatccaggttcttgcttcaaagtctgccatcttggaagccacacccaaggaggtgcctatggtggctgtgcctccagtgggctctaaggccagttctcctgccaccagcagtcagggcaagaagggacagggagcccagaaccaggccaagaagggggagggagcccagaaccaaggcaagaagggagagggggcccagaaccaagctaagaaaggagagggggcccagaaccaggccaagaagggggagggggcccagaaccaaggcaagaagggggagggagcccagaaccaggccaagaagggggaaggaggccagaaccaggccaagaagggggagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggggagggagcccagaaccaggctaagaagggggagggggctcagaaccaagctaagaagggtgagggggcccagaaccaaggcaagaagggagagggggcccagaaccagtccaagaaaggagagggggctcagaaccaggccaagaagggggaagggggccaaaaccaggccaagaagggagagggggctcagaaccaggccaagaagggggagggagcccagaaccaagctaagaagggggagggggtccagaaccaggccaagaagggggtagaaggggcccaaaatcagggcaaaaagggggaagcaaaccaaaaccaggctaagaagggtgagggaggccagaaccaaaccaagaagggggaaggaccccagaaccaaggcaagaagggagaagcagctcaaaaacaggacaaaaaaatagaaggagcccagaatcaaggcaagaagccagagggaacctcaaaccaaggcaagaagggggagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagagggggcccaaaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagaggggccccagaaccaggccaagaaaggagagggggcccagaaccagggcaagaaaggagagggggcccagaaccagggcaagaaaggagagggggcccagaaccagggcaaaaaggcagagggggttcaaagtcagagcaagaaaggggaagggacccagaatcagggcaaaaagggggatggaaatcccaaccaaggcaagaagggggagggggcttccaaccagaacagaaagacagatacagttgctaatcagggcacaaagcaagagggtgtctcaaaccaggtgaaaaagtcagaagggtcccctaatcaaggtaaaaaggcagagggagccccaaaccaaggcaaaaagaaagatggatcccccagccaggccaaaaaagtggatgcagctgccaatcagggtaaaaagtcagagatggctcctgcccagggtcaaaaggcaagcatggtccagagccaggaggcaccaaagcaagacgcacctgcaaagaagaagtctggttccaggaaaaaaggagagcctggtgagtgcccgtctgctctgtgagcttgagggaaagccgctgactgtgactga^TGGACTTCCTTAGTGCTCTGCTGTTTGCCATTGTCTTTTCACCCCAGATAGGGTATTGATGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGAGAAATAACTCCACCTAGTCTCTCTTGGACTATTGAGCTTTTGGGATTTTATACAGGAGCA
This mutation is a 2404 bp deletion beginning at Chromosome 2 position 143,988,095 bp and ending after 143,990,498 bp (GRCm38/mm10). In addition, there is an 11 bp insertion at the deletion site (TTAGTGCTTAG).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46160 | AAA AAG GAG AGC CTG GTG AG | Wild type Forward | A | |||
46161 | CAA ACA GCA GAG CAC TAA GGA | Common | A | |||
46162 | AGG GGG CAC CTT GAA CTT G | Mutant Forward | A | |||
46163 | Fluorophore-1 | CGT CTG CTC TGT GAG CTT GA | Quencher-1 | WT Probe | ||
46164 | Fluorophore-2 | AAC TTG AGT CGT TCC CTT AGT GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46160 | 0.40 uM |
46161 | 0.40 uM |
46162 | 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 |