Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:134605508+134605607 100bp CTCATGACTATGCAGCGC CCCGCCTCATTCTTTCAAAC
Mut= 95 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
X-Linked
Wt Sequence (deletion in lower case):
ATATGGCTCCTTTGAAAAACTTACTGATGTGAACTTGTAAAATAATAGTTTTCTTTGTTTCTCTTACTATATTATATACACTTTTTCATGACAATAGTTTTCTAATTTTTTTAAAGCCACATTGAATTTGCATTGAGCCAAGCTTGCCACCAAGAGTCCATCAGTCACCatgatgctgagcacagagggcagggaggggttcgtggtgaaggtcaggggcctaccctggtcctgctcagccgaggaagtgatgcgcttcttctctgattgcaaaatccaaaatggcacatcaggtgttcgtttcatctacaccagagaaggcagaccgagtggtgaagcatttgtcgaacttgaatctgaagatgaagtgaaattggctttaaagaaagacagagaaaccatgggacacagatatgttgaagtattcaaatccaacagtgttgaaatggattgggtgttaaagcatacaggtccaaatagtcctgatactgccaatgatggttttgtacgactcagaggactcccatttggctgtagcaaggaagaaattgttcagttcttttcagggttggaaattgtgccaaatgggatgacactgccagtggactttcaggggcggagcacaggggaagcttttgtgcagtttgcttcacaggagatagctgagaaggccttaaagaaacacaaggaaagaatagggcataggtacatcgaaatcttcaagagtagccgagctgaagtccgaacccactatgatccacctagaaagctcatgactatgcagcgcccgggtccttacgataggccaggggctggaagagggtataatagcattggcagaggagccgggtttgaaagaatgaggcggggtgcctatggtggagggtatggaggctatgatgactatggaggttataatgacgggtatggttttgggtctgatagatttggaagagacctaaactactgtttctcaggaatgtctgatcatagatatggagatggtgggtccagtttccagagcaccacggggcactgtgtacatatgagggggttaccatacagagccaccgagaatgatatttacaattttttctcacctcttaatcccatgagagtgcacattgaaataggacctgatggaagagttactggtgaggcagatgttgagtttgctactcatgaagacgctgtggcagctatggcaaaagataaagcaaatatgcaacacagatatgtggagctcttcttgaattctaccgcagggacaagtggaggagcttatgatcatagctatgtagagctctttttgaattctacagcaggggcaagtggtggtgcttacggtagccagatgatgggagggatgggtttatccaaccagtctagttatggaggtccggccagccagcagctgagtggtggctatggagggggttatggtggtcagagcagtatgagtggatatgaccaagttcttcaggaaaactcaagtgactatcagtcaaaccttgcttaggaagcaagggaccatccaataacatctacatgaataaaagctatctttataggaactggatagagtagggagggtgtctttatatccagtatgattggtaaATGGGAAGTACAACTGCTTCTGATCACTATTGGTCAGCTTCTTTCTTCTTTTTCCTTTTTTTTTTTTTAAAAAAAAGAACAAAAATTTAAGTTTTAACAGTTTCGCATTACAAGCTTGTGATTCATGCTTACTGTAAAGTGAAAGTTAAGATTGTTTTCAAACTTTAAGCTCAGCAATTTTGAACACTGAAAATATTCATCTAGGATGTAATAACAAGTTCAGTATTG
This mutation is a 1451 bp deletion beginning at Chromosome X position 134,604,908 bp and ending after 134,606,358 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39357 | CTC ATG ACT ATG CAG CGC | Wild type Forward | A | |||
39358 | CCC GCC TCA TTC TTT CAA AC | Wild type Reverse | A | |||
39359 | TGA ATT TGC ATT GAG CCA AG | Mutant Forward | A | |||
39360 | GAA GAA AGA AGC TGA CCA ATA GTG A | Mutant Reverse | A | |||
39361 | Fluorophore-1 | AGA GGG TAT AAT AGC ATT GGC AGA | Quencher-1 | WT Probe | ||
39362 | Fluorophore-2 | ATC AGT CAC CAT GGG AAG TAC AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39357 | 0.40 uM |
39358 | 0.40 uM |
39359 | 0.40 uM |
39360 | 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 |