Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.
Mutant= 90 bp
Wild Type = 103 bp
Wt Sequence (deletions in UPPER case):
gcgaagggatgaagatctccttggagtctcaagagtaccagagtcagccctgaatcttgtagacacactgacaaggatgtctagcccaaatggatgtacatttgtgtgctcagtgtatctgtgtgtgaatgaagccacttggattctggggtgggcaaagaagaccttacagagtctagagcagaaggcctgtgttgccaccaaaactccttcccctggtatcactatacccaccttgtactctgttcaggaggctgcccatggagggcagatgaaggctcccaccacccgatgcagttgagtcccatcctgccctctctgcccacattggcttcctgctGCTATTCTAGTGCCCACAATAAGCTGTTCCCACCAAAAAAAAAAAAAAAAAAAAAAAAAAAACTCTTTGGCAATTTTAACCAAAGTGGGAAAAGTGATGTAAGGCATATTCTATCAACAAAAATTGCTACTTACTCTCCCAAACTCCTGGAGAACAGCTTTGGTAGCAATATCATGGGAACTTGTGTCAGCCAGGTCAAGAGGCAAAACCAGTATGTCGTTTTCTCTCATGTTGCCATTCTCTGAATTAAGACACATCAGCACATTAGTGATAAAGTCACGCTACAAGCCAATCAAGCTCTTTCCTGCTCTTACTAAGTCGATCCCTGCATCTTTCcaaaggcatggagagacgtagtgggactcatgaaagaactctggcaaaaccaagattattctaccgcaaacaaaacagaacaaaccacaaaaacaaaaaaatgcacaatagaaacccacagctcatttattagtatagctacatttgtcttgaaatatgctcatgaggctggcgagatggctcagacggtaagagcactgactgctcttccgaaggtcctgagttcaaatcccagaaaccacatggtggctaacaaccacctgtaatgagatctgatgccctcttctggtgcgtctgaagacagctacagtgtacttatgtataataataaataaatctctgggccag
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GTGGGCACTAGAATAGCAGC and CGATCCCTGCATCTTTCCAA. This resulted in a 336 bp deletion of Chr12:72,621,714-72,622,049 (GRCm38/mm10) that removes exon ENSMUSE00001380275.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
77043 | GAT AAA GTC ACG CTA CAA GC | Wild type Forward | A | |||
77044 | CTT TCA TGA GTC CCA CTA CG | Common | A | |||
77045 | ACC CGA TGC AGT TGA GTC | Mutant Forward | A | |||
77046 | Fluorophore-1 | CAA TCA AGC TCT TTC CTG C | Quencher-1 | WT Probe | ||
77047 | Fluorophore-2 | CCC TCT CTG CCC ACA TTG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
77043 | 0.50 uM |
77044 | 0.50 uM |
77045 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
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 |
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