Protocol 50180: Probe Assay - Dhrs7l<em1(IMPC)J>
Version 1.0

Notes

Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant=  90 bp

Wild Type = 103 bp

Sequence

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.

JAX Protocol

Protocol Primers

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

Reaction A

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

Cycling

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
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.