Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:58493780-58493931 152bp AAAAACATTCTGGGAAGACATC TGTGATCAACAGAGAGCAAAGT
Mut= 147 bp
Wt= 152 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCAGAAGGATTTCTGAGTTTGAGGCCAGCCTGGTCTACAGAGTGAGTTCCAGGTCAGCCAGGGCTACACAGAGAAATCCTGTCTGGAAAAAAACAAAAAAAACAAACAAAAAAGATAAAAACATTCTGGGAAGACATCTTAACGGCTTTTTTTAAAAGTTGTACTCTTAATGTAggtaggtttatatacctttctgataaaagtagtacaggtgtttgttttaaggagttcatataagtgaaagctactttgctctctgttgatcacaaaagtgcttttatttttccccgattacagctttagaactagcaaaaacatcaggaaaaaaggatccacttgactcaaatgatcctgaaatcacaaaaaacattgctcaaaaaagtaaaggtaagataccatcaggtgatctgtgtgtgctacttttttttccttaaatctaagtgaatttccatgtattttttttgttttcagaaaatgtatttaacagaatgcaatttttgttttgttttgttttgttttgggtttttttggtgttttcaagacagggtttctctgtatagccctggctgtcctggaactcactctgtagaccaggctggcctcaaactcagaaatccgcctgcctctgcctcccgagtgctgggattaaaggcgttgccatcactgcctggccagaatgcaatttttaatatatctatacatatcagtcaataaagggcagaaagtcatctgtgccaccctatgttgggagaattgacgaggaaacctaaaacaatcatcaagtcctataatcatttgtctgagttagccagtttctcatgcaaggtttggaagttgaatggcttgttgcaaattgcagagcatctatttttgaattacccattccagataagagagacagtgtgctttctggtccaaatgaaaaaggctcggggttttgcttccctgttagaatgccacagaaattcaagaatgctgaaaatgttgccactggttagagtagccatataattcttggtgaccatttagcctgtagttaaatggaaactcagtacaatacagtggcttgagtgtaaaacgtgcagctttgctggcaacaaaactatgtcatgatctaagatgaaatatttgtagtaaatgtgacctttgttccgtttagggcattctacttcagagaaagctccactggtggtaagtaactgacaaaacaagcaagtgtgataaatggtagctttctgtcttcgtttcagtgttaaagtttgtgccagttatatacctgttatctgaggttcataaaggacagatgacttacagagtacctcacttggaataaaactggagaatgaaccctggtttcccCTACATagctgttctctgctcttcctgctccttcacagctatcctgcgtatttgtaacttagtagacctgacctcggaatTTGATGAGTTTGGTTTTGTATTTATAATGTTCTCTTCTTAGAGTATTTCGTCCATTAAAGTCTATATTGGCTTGGGTAGGTTCATTCCAGTTTAGCCAAAAGTAATCCACAGTTTTTGTCTCCTTTGTGTTGTAGTGATTTTG
This mutation is a 1259 bp deletion beginning at Chromosome 1 position 58,492,609 bp. There is a 6 bp endogenous retention (CTACAT) after 58,493,784 followed by 74 bp of the deletion and ending after 58,493,873 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46346 | AAA AAC ATT CTG GGA AGA CAT C | Common | A | |||
46348 | TGT GAT CAA CAG AGA GCA AAG T | Wild type Reverse | A | |||
46349 | GAA CCT ACC CAA GCC AAT ATA GA | Mutant Reverse | A | |||
46351 | Fluorophore-1 | AGT AGT ACA GGT GTT TGT TTT AAG GAG TTC | Quencher-1 | WT Probe | ||
46352 | Fluorophore-2 | CTC TTA ATG TAC TAC ATT TGA TGA GTT TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46346 | 0.40 uM |
46348 | 0.40 uM |
46349 | 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 |