Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 110 bp
Wild Type = 128 bp
chr1:55148831+55148958 128bp TCCCAAAGTTTCTACCACTCC CAACTACACCCACTACATTCCAC
Wt Sequence (deletions in lower case):
TGTATGTAATTGTACATAGAAAACACCTACATAATTTTTATATAACTAAAACATCTTTCATGTTTAATCATACATATCAACAACTTCCTATCCTTCAGTTGATCATAATCAGAAGTCTTACCACAtatacacggttaatgtgaacacctttgtaacataactgtatacagttctgaaaactgcattacaatagattctcaatcaaaaagtacttgtttttctagagtcttggtgaaatttctaagtactatccagaaaaattaaacagatttttattgtataaaaggataaccagcttgaccaccttaaaaaataaaagaagaaaatacacaaagcctggataataaatgtgctttttgcatttttaggcaattctgccttgaactaaatggacttgctgtcaaacttcaggtagtattttctttatgttaaagctgtaattcaaagttctcatttttaaaaatgattttgtatgcttagatttttccagtgtttcctattatcctttttatattttatatagaaatattataacccaaatgaccattttttccattatcattctacatattaaaaaaataaaattatttttaacagagtgaatgccatccagatacctgtactcagatgacagcaactgaacaatggatttttctttgtgcagctcataaaactccaaaagaggtgagaattgagagcttccagtacctgctcattggcaccattactttgggcctgaagtgaggccaaacatctcagatgggagcatttagggaagcaagtctgcttacatggcatccaagaaagagagtgaagagtcaggagcccagtgccctcaaagacactgcccatggcctgcttcctccaactcggcctcatctcccaaagtttctaccactcctgagagactggccagctgggacccacatcttcaggagataagcccTTGGGGAACATTCCATATCCATATATGAACAGAAATATTGTGGAATGTAGTGGGTGTAGTTGTACGATATTATACTTAACACTGCTGAATTTGTTTAATATCTGTGGGTGTGATGTATGTGCAGTTGTGTGTACATGTGTGTACATGCACCCGTGGAGATGAGAGAAGTCCATA
This is a 821 bp deletion beginning at Chromosome 1 position 55,187,235 bp and ending after 55,188,055 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
60215 | TCC CAA AGT TTC TAC CAC TCC | Wild type Forward | A | |||
60216 | CAA CTA CAC CCA CTA CAT TCC AC | Common | A | |||
60217 | CAA CAA CTT CCT ATC CTT CAG TTG | Mutant Forward | A | |||
60218 | Fluorophore-1 | AGT CTT ACC ACA TTG GGG AAC ATT C | Quencher-1 | MUT Probe | ||
60219 | Fluorophore-2 | AGC TGG GAC CCA CAT CTT CAG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
60215 | 0.40 uM |
60216 | 0.40 uM |
60217 | 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 |