Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:127796380+127796470 91bp CCCCTACACCATTGTACTGTGA GCCCTTTCCTCTTGCTTTGT
Mutant= 107 bp
Wild Type = 91 bp
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):
GTCCAGGCTGGGCTTGAACTAACGATCTTTCTGCCTTAACTTCTCACCACCACATACTTGATTTAATTTTTAATTACAACCATCATATTTTTTTGTTTACATCACTATTTAAATGAGAAAGGAGTGAGACTTCCCTCTGCTTCCCCTACACCATTGTACTGTGAGCAATCTCATAACcgtccacattataagactgggcaaaggagaaatgaaacaaagcaagaggaaagggctagtattgttcttactataggatcccctaccgcattttgagcacactctttgagagggcagtacaggctgaactaaccgcatgctttgaccattgcaggccggggagcgtcatgtccacctttgaagacgctgatacagaggagacggtcacttgtctccagatgaccatttaccatcctggccaacaaagtgggatatttaaatcaataaggttttgcagcaaagagaagtttccttccattgaagtggtgaaatttggacgcaattccaacatgtgccagtatacgtttcaggacaaacaggtgtcccgaattcagtttgttttacagccgtttaaacagttcaacagctccgttctctcgtttgaaataaaaaacatgagcaagaaaaccagtttgatggtagacaaccaggagctcggctacctcaataaaatggacctgccttacaagtgtatgctcaggttcggagagtatcagttcctgttgcagaaggaagacggagagtcggtggaatcttttgagactcaatttatcatgtcttcaagacctctcttgcaagaaaacaactggccaacacagaatcccataccagaggatgggatgtattcttcatacttcacccacagaagttctccttcagaaatggatgaaaacgaactgtgaagagggtccaactggagacacattgaaggatgaggacacatgggtcggatgtcaagagacatcctacttccgagtttgtgagtgtagcgtagcgcggctgtcctcatgctgactttcgttttggtaatagcatttggaagtctctagactgtgttaatcatcaacttagtcaactgagtttcggctctacaaagaattaagtgtacatctgtaagggttggtgcatcagacacgtcttctgggtaatgaggtcacccttgttgcttttctgcatgatgttacccccatgctttgtcttggtggcagccatctcttggcccggtcacatcatttcgtagcagcctttgtttttcaggtttagagctcgggcagattgctcactggtgtctgtggcgtgctagcgcttgtagaactagagtcctggaataagttctagagtgctgagtcactgagtcaccatggcttccttatggaaagacttgggaaatagctccttgattttctttctgtggaacggtagtgtcgctttcctatatgtaggacctacaacaaacatttaaagaacactgagatgaagatggttttcttacaatattgaaagtgaattttatgtatctcacagatttaaaaatggcagaaatcaaaacttttaacagcctctttgcacatgataaagccggagcccagttccttagttgcttctttggaacttcttaaaggaaaacatgtattcttaaaggaaaacatctattcttaggctgccctatagaagtcagtacctgtgaatatttatattaaatgcttaattatttctaaaattttagtttcacataaagttgtatttatttaaaagattctcattcacttcattttggctagattaagatgaatgttagtgaacattatgtaaaagaggatgaaagccattaagttaagataaattctagcattactagtaagtaaggcaccctgtatagcttcctctgtaaatgaaatttaatgctgtaacaggtacaggattttgggtaggggaggaggtcaggtgggggaagttagccacattcatattttgtttttgtttttgtttttgtttttgtttttgttttccaacaatagcttgctttgaagctcaggctggcttggaactcttgatcctcatacatcggccccctgaatgctgtgcctagcttaatgtaactgtatttctgcaacagccctttgaaattatttctaataaactgtttggcctagtaccatgtgaatgttatctgcagtatgcctcttgctgcagatcacCAGTCAGTCACCCCTTGCCTATAAAATAAGACATCAGCTGCTGCCTCAGCAGGGATTGCTGCTATAGGCAACCCTTTATGTGCTAAGTGTCCAGGTGAGTATTTCATTTCTTGCCTTGTTTTCCTGCTGACTCCCGTGGTAAGCTGTTGTGTGTAGCTTTTCACTATCCTAGCTATGCTCTGGGAATCAGACTGAACTACCAGCCCCCTCCCAGCTTCCCTTGAATCCATGGATA
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences GCCCAGTCTTATAATGTGGA and GACTGGTGATCTGCAGCAAG, which resulted in a 2018 bp deletion beginning at Chromosome 3 position 127,796,415 bp and ending after 127,798,432 bp (GRCm38/mm10). This mutation deletes ENSMUSE00001352154 (exon 3) and 197 bp of flanking intronic sequence including the splice acceptor and start of translation and is predicted to result in a null allele.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46353 | CCC CTA CAC CAT TGT ACT GTG A | Common | A | |||
46354 | GCC CTT TCC TCT TGC TTT GT | Wild type Reverse | A | |||
46355 | GTT GCC TAT AGC AGC AAT CC | Mutant Reverse | A | |||
46356 | Fluorophore-1 | CCA CAT TAT AAG ACT GGG CAA AG | Quencher-1 | WT Probe | ||
46357 | Fluorophore-2 | CAT AAC CAG TCA GTC ACC CCT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46353 | 0.40 uM |
46354 | 0.40 uM |
46355 | 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 |