Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 100 bp
Wild Type = 98 bp
>chrX:74152402-74152499 98bp GGGTTACTCACTGCCAGCAT TCACCTGTTTTTCCATCAGTG
Wt Sequence (deletions in lower case):
TGGGTTGAGATTTAATTTGCATGTCATGAAACAATAAGCCCTACTAGCAGTCATTCCTTGTCACTCCTGGTTCCCAGTTCCTGGCACCCACTCATATGACTTGTTTGTGGATTGCATACAAATGGAACTTTACACAATGAGGTCTTTATAATAAATATTCATTTATGGAGTAGATACTGCATCTTCAACACTGCTCCTGGGAGCATTTGGGGGGATAGCTTAAAATTTAATTTCTATACTTTCTAGTCTATTGCACACTTGGTAAAATAGTAGAGAATAATTGTTAATAAGATCTATTAGTGAAAGTAGTGTAAGTATTCCCAGGCAGAGATAGAAGATCCAGCCCCCTTTGACAGAATCCTAGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTTTGTGTAGGGATGTTGTTAGTAAATCTAGCTTCGGGTTACTCACTGCCAGCATGAGCTCTGAGTAAAggtacagagggagtgttactttctctcttgcatcctagacaatcactgatggaaaaacaggtgaatgatcacttgcagagatacctggatgagatttgccacctcaaacagcatctggcatgcactgaagagaaaatggcctatttgtcctatgaaagagccaaggaaatatgggtgagggcactggggagaaaggactagtgagaaggatatggggatgaatagatgtatttggatatgcccaagccaagacaagagaagatatagagctgtgtctggcctgacagtacaatagtaacctggtttagagccagcagtgtccaccctgcatgcctgtgtgaactccactccttaccatgcagccaaggtgctttggagactttggacagtggttctaatcttgtagggtcaattactgactcataccctggaatctgtgtcctataaatcaggagtgaggcctgacaaacccaaggaatgctggggtgtttgcattgctagtagagagaaAGCCTTTGGGAATCTCTTCTTGGGGTCTAGCTTTTTGCCCCTCAGTCTTTTCCATACTCATAGCTCCTACTCCTGTTCCTGGTTAGACCTTTTTCACCTAGGTTTTCAGTCTCAGCAGTAGATAGTTCTCACCAGGACCCATCAGCCCAGCATTCCACAGCAATAGTCTGTCTCTGCTGGGCATACCCAAGAGGTTCTTCCCATCTTTTCTAAAAAATTCAACTCATCAGGTATGATGGCACATGCCTTTAATTTCAGGACTTGGGAGGCAGAGGCAGGCAGATCTCTGAGTTTGAGAACAGCCTGGTCTATCTAACAAGTTTTGGCTCACCTGGAACT
This mutation is a 510 bp deletion beginning at Chromosome X position 74,151,956 bp and ending after 74,152,465 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44441 | GGG TTA CTC ACT GCC AGC AT | Common | A | |||
44442 | TCA CCT GTT TTT CCA TCA GTG | Wild type Reverse | A | |||
44443 | GAG CTA TGA GTA TGG AAA AGA CTG A | Mutant Reverse | A | |||
44444 | Fluorophore-1 | AGG TAC AGA GGG AGT GTT ACT TTC TC | Quencher-1 | WT Probe | ||
44445 | Fluorophore-2 | AGC CTT TGG GAA TCT CTT CTT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44441 | 0.40 uM |
44442 | 0.40 uM |
44443 | 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 |