Protocol 48770: Probe Assay - Uprt<em1(IMPC)J> ALT 1
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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=  86 bp

Wild Type = 119 bp

>chrX:104498118+104498236 119bp CAACAAGCAATCTGGCATTT AGACTCTCCTGCTATCATGT

X-Linked

Sequence

Deletion in lower case:

TGATTTTGTTACTACCAATGGTGTTTTGTTTACCATAACTCAAGTTGAAGTTATGTGCCTTTCATACTCTTGACTGAATCAGAAGGCACACTGGTGCCATTTGGTGTCAGTCACGTGTCAGATGGAGGTTGGATACTTAGCAACAAGCAATCTGGCATTTAGATCTGTCCCTGCAGTTAATACATGGtgggggacccacttcctacctccctaacctaagccctgagatcttgagtctaacatgatagcaggagagtctctgcaagctctgaacttcttctttctttgaccttctagatcagacttgttgtagaagagggactgaatcagctgccatataaagaatgtatggtgaccactccgacaggtaaccagggcttgtactcgcaaattttcctcataaaaattctgagctagactggggttagaGGAGGGAAATGGAAGGAAGACATCCTCTGTAAAATCAGTGTGAAAAATCAGGAGTTTATGTATGACTGGGTTTTTTTTTTAACATCTTTAAAAACTAAGAGAGTTTTTGTTAAGAAATCTACATAGGTGACTTTTTGTTAATGGCTTGCTTGTCAATTCAAGTTAAAGGTTATTACATACTCTTCAGCATGCAGGTGTTGCTTCTGATGGC

This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: CTGCAGTTAATACATGGTGG and GCTAGACTGGGGTTAGAGGA. This resulted in a 242 bp deletion of ChrX:104,498,165-104,498,406 (GRCm38/mm10) that removes exon ENSMUSE00000208388.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
72993 AGA CTC TCC TGC TAT CAT GT Wild type Reverse A
72994 Fluorophore-1 CCT AAC CTA AGC CCT GAG AT Quencher-1 WT Probe
74069 CAA CAA GCA ATC TGG CAT TT Common A
74070 ACT GAT TTT ACA GAG GAT GTC T Mutant Reverse A
74071 Fluorophore-2 TCC TTC CAT TTC CCT CCC CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
72993 0.40 uM
74069 0.40 uM
74070 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
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.