Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:140610610-140610704 95bp TAGAGAGGGGCCAAGGAAAG CCCAGGAACTGAGCCTATTC
Mutant= 100 bp
Wild Type = 95 bp
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):
TCTGACTGAGGTTGGGCCATGGGGGCGGTGTGGGGCAGGCAGGACAGGTAGGGAACTTACTCACTAGAGAGGGGCCAAGGAAAGGCAAGGGATTCTATAGGCACCCTGgttccttcgaggggcggggaacagggggtgggaataggctcagttcctggggccaacctctgtctcacaagctctctctctttgctcttcccacagacctggacccagcagctgcaccaccccagacagagtaagtgagctttgtccactgggaaaagagtagagcccaacaggggcgtgggacagggtaggacagtgactagagaatactgttgtttgttaagaccctttgcctggacgaggttggccctgaggctcgctgtggtggcctctttttctcttttgcctgggagcattgcattgcattgcctggtcccagacccacattcaggcctctccctacccatgtggcctcagtggactcacagactctcccagatttcctgtgttctgacttgaaacggaagggagagGAGACTCGCTTCCCTGGAGTTATCCCTGGGAACTAAGATGCTGTGGGATTGCTTCTGCTGATACCAGTCCTTCTCTTTCAAATTTTTAAATTACAAAATTTTATATTTATTGTGTGTGTGAGCATGCACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGAGTGTTCGAGTG
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences TGTTCTGACTTGAAACGGAA and CCCGCCCCTCGAAGGAACCA, which resulted in a 413 bp deletion beginning at Chromosome 4 position 140,610,248 bp and ending after 140,610,660 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000335941 (exon 4) and 379 bp of flanking intronic sequence including the splice acceptor and donor and is predicted to cause a change of amino acid sequence after residue 74 and early truncation 43 amino acids later.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46646 | TAG AGA GGG GCC AAG GAA AG | Common | A | |||
46647 | CCC AGG AAC TGA GCC TAT TC | Wild type Reverse | A | |||
46648 | AGA AGC AAT CCC ACA GCA TC | Mutant Reverse | A | |||
46649 | Fluorophore-1 | TTC CTT CGA GGG GCG | Quencher-1 | WT Probe | ||
46651 | Fluorophore-2 | CAC CCT GGA GAC TCG CTT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46646 | 0.40 uM |
46647 | 0.40 uM |
46648 | 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 |