Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:115280591-115280698 108bp CCTTAAACCTTGAAAACTGGTTC TGGCTACACATATCTCACTATGAAG
Mut= 121 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCTTGATTTCTTAACTTTTTCATACGTTGTTACCTTGTAATTTGCCCTTCACTTTTGTCTGTTAAAATAACCTGCTTATGATTTAACTGGATAATCACAGTTTTCCTTAAACCTTGAAAACTGGTTCTTTTATCATCTGTTCATCACAGTAgtagcctcttcacagggacttgcttcacagttctctcttcatagtgagatatgtgtagccatatattgaatataaagaaaggtgtccttgtgttaactctttttataacatttcctcttctcttttgtccaggtttaactatagatcaacacatcatcttgcatctcatggattctatgagtttctaaattggtttgatgaaagagcatggtacccactgggaagaatagtgggtggcaccgtaagtatattccttacctttgcggtaactgtaaactgtggagtaaagcatttcctgctaagggacgagggtctcggaaggccgtcattagttcacagacacatcttccatggctaattagccagtgtttctcagctccgctcttggcagtttatttgtaggatatcttacactgccccccatttctgattgaggtcaggggctggattaagaatctgctgtcacaaagtacctcaagtccagagtgcttttcgctttgtttctgaacaattgagtttaaaagcttttgggggaaggaaataaaaattttgaaaatcaaattaattaaatgtttccagcctcaagcctctgaattaatgtagataattgaaccgtattgagtagggtagagtGTTCTGCCTCCAGAAGGATGACATGAAGGTTCTTAGGGTTCAGTGTGTAAGGAGAACTCCTAGAGAAGGGGGAAGATGTGGCAGGCAGCTCTCGGAGAAGTTTGTACACTTTCGTTTCTTCTTGAATTT
This mutation is a 633 bp deletion beginning at Chromosome 9 position 115,280,019 bp and ending after 115,280,651 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41319 | CCT TAA ACC TTG AAA ACT GGT TC | Common | A | |||
41320 | TGG CTA CAC ATA TCT CAC TAT GAA G | Wild type Reverse | A | |||
41321 | TTC CCC CTT CTC TAG GAG TTC | Mutant Reverse | A | |||
41322 | Fluorophore-1 | CAC AGG GAC TTG CTT CAC AGT | Quencher-1 | WT Probe | ||
41323 | Fluorophore-2 | CAT CAC AGT AGT TCT GCC TCC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41319 | 0.40 uM |
41320 | 0.40 uM |
41321 | 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 |