Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:31160218-31160309 92bp GCAGAACATTGACTCGTCCT GCGCCTCTCCTCACAGAGAT
Mut= 92 bp
Wt= 92 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTGTCTCAGCTCCAGCTCAGGGCATCTGATGCCTCTGGCTTCTGTGTGTACTCATGTAATTGTGCACGTACCCACCTACATACATGTGCTTGCAGATGAATGTTAAAGCTAGCCTACTCATTTTACGATCTGTGCTACTGTGTTATTGTCACGTTTACataaaggataacttttaaccatttgtatctgtgttctcacttagaatgaaaatctgtttctcaagagtaaagagacctttaaagaaatgaactaacttgtctactatcccattctcctagggctgctttggaagaggtagaaggagatgtggctgagctggaactaaaactcgacaaggtaagctttccctttaagtctgcagaacattgactcgtcctttccgtgatgagcggttagcGCCCACCACAGTGGCTGGTTTATGTACCTGAGATCTCTGTGAGGAGAGGCGCTGTCATCCTGGCTTGCTGCATGAGGAAATGAAGGCTAGATATGTTGAGCAGCTTGCAGGTTCCAAGATGAGTTGTCAAAACTGGTGTCCGCGCCTCACTTCAGAACCT
This mutation is a 239 bp deletion beginning at Chromosome 16 position 31,160,270 bp and ending after 31,160,508 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39471 | GCA GAA CAT TGA CTC GTC CT | Wild type Forward | A | |||
39472 | GCG CCT CTC CTC ACA GAG AT | Common | A | |||
39473 | CAT TTT ACG ATC TGT GCT ACT GTG | Mutant Forward | A | |||
39474 | Fluorophore-1 | TTC CGT GAT GAG CGG TTA G | Quencher-1 | WT Probe | ||
39475 | Fluorophore-2 | ATT GTC ACG TTT ACG CCC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39471 | 0.40 uM |
39472 | 0.40 uM |
39473 | 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 |