Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:162933755+162933873 119bp TGCTTTGGATAAACTGGATGG GCCCTCGTTACCTGGATCTAC
Mut= 126 bp
Wt= 119 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
CGGAAGCCATGACGACAGCAGCCTTTTGCCATTAGACGCAGGGTGATGGTGAGGATTCCAAGGGTTAGACAAAACTGGTTAATCTGAACTAGGTGACTGTTACCTTGCGTGTTTTGTGGCCAAACCACCACCAAAAACCTCACACTGTGATGTAAGTACTTAGTGTAACTAGTAAACATTTTTGTAAAATGTAGAAATGCATGTAATCAGTTAAGTTTTATATTTTACAATGTTCTGTAAAATAAAACTTAGCGAGGTAAATTGAATAAAGGAGCAGTCACTCTCTAACAGattgtaggagaggtttagttggatttagtctatttgacttgcccttaatttaatttgtggcaaatcacaaatgtatcgagggtttagcagtataatagcaaagtcctactccagtaataaacgttgctatgtttgtactaactttcaaaaacatgcatgcctgtcattgcaacgcatctaattactcccttcatgtgataaaggtggtggaggtggatacagcagtcggagaacttctggcagagacaaatatggaccacctgttcgtacagagtacaggcttattgtagaaaatctgtctagtcgttgcagttggcaagacttaaaggtatgtacctggggttttaaagacagagcagttactttccttaagagttggctacgtggtcttgctttagatttctggagcttatcagtcttaactgttttaggatttcatgcggcaagcaggagaagtgacttatgcagatgctcacaaagaacgaacaaatgagggtgtgattgaatttagatcctactccgacatgaagcgtgctttggataaactggatggtacagaaataaatggcagaaatattaggcttattgaagataagccaagaacaagccataggcgctcctactctggcagtagatccaggtaacgagggctggggggaggcttgtcatttccagttgttgcctagttgtatttacggtgtttcttggttgtagtaagcagatattctagccacagATGGGTAGTTAAAACATATTCCTTTTTTTTTTTTAAGATTTATTTATATATTTATTTATTATATGTGAGTGCACTGTAGCTGTCTTCAGACACTCCAGAAGAGGGCATCAGATTTTGTAACAGATGGTTGTGAGCCACCATGTGGTTGCTGGGATTTGAACTCAGGACCTTCAGAAGAGCAGTCGGTGCTCTTAACCTCTGAGCCATCTCACCAGCCCCCTAAAACATATTCTTGAAAAGATTAAAACATCACTGTCTCTTGTGTGTAGATCACG
This mutation is a 736 bp deletion beginning at Chromosome 2 positive strand position 162,933,223 bp and ending after 162,933,958 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38126 | TGC TTT GGA TAA ACT GGA TGG | Wild type Forward | A | |||
38127 | GCC CTC GTT ACC TGG ATC TAC | Wild type Reverse | A | |||
38128 | AGA AAT GCA TGT AAT CAG TTA AGT TTT | Mutant Forward | A | |||
38129 | AAA AAG GAA TAT GTT TTA ACT ACC C | Mutant Reverse | A | |||
38131 | Fluorophore-1 | AAA GGA GCA GTC ACT CTC TAA CAG AT | Quencher-1 | MUT Probe | ||
39197 | Fluorophore-2 | AGA ACA AGC CAT AGG CGC TCC TA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38126 | 0.40 uM |
38127 | 0.40 uM |
38128 | 0.40 uM |
38129 | 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 |