Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 113 bp
Wild Type = 117 bp
>chrX:60295009-60295125 117bp AGCAAAGAAATACCCACTGCT TCTTAAGCTGGTAATGAGCAAGG
Wt Sequence (deleted region in lower case):
CAGTTTCAAAATCATTACTTAAATTGTTTGTATAAGAAAAATCTGTTTGTGTAATGGAACCATTAATGATTTCTTCAAATCTTCATGGGGATTTCAGTTAACTATATGCATGCTACAAATGCAATACTCTCAACTGTCAGTTTCAAGGAAAGTTTTAAGTTGTTTTTCTTTTCCCTttgaaatccttttgaggcagagataattatctagttgttggtagtttaaaatagtatcctatatttggtgggtaatatccatcaacatttgtaatattcgctgatccctattactgttaaacattgctgtttctggaaccagttttatatagttataaaaaaagaaataatttatatttgattatcttctgctgtcatatagacacattatgcagtacgagataccattgcactgtgtacagccgaatccattgataatctccgagcaacaattgaatgtgagcagcctcaacctgatctctacaggtaagaagaccttgcttcaccttacttatgtctgttgtctctgcttctttttcctaccagtgtagcaaagaaatacccactgcttatttaccagcttgctctttgtatgtttttataagctgttcttattccaaggAAGGGAGATGAATCAAATTAGCCTTGCTCATTACCAGCTTAAGATAAATCATTATGGACCCAAGCGTCCTATGTTTTTCTTTTCTTTTAGTTACTTGGATCAGTTTTTTAGATGGTTAACCTAAGCTTCATCTATTAGACTAATAGAGTATCTAGAGTTAATTAAAACAAAAA
442 bp deletion beginning at Chromosome X negative strand position 60,295,494 bp and ending after 60,295,053 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38525 | GCA TGC TAC AAA TGC AAT ACT C | Common | A | |||
38526 | TCT TAA GCT GGT AAT GAG CAA GG | Mutant Reverse | A | |||
38527 | AGC AAA GAA ATA CCC ACT GCT | Wild type Reverse | A | |||
38529 | Fluorophore-1 | CAA CTG TCA GTT TCA AGG AAA GTT T | Quencher-1 | MUT Probe | ||
38530 | Fluorophore-2 | CCA GCT TGC TCT TTG TAT GTT TT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38525 | 0.40 uM |
38526 | 0.40 uM |
38527 | 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 |