Protocol 43931: Probe Assay - Ly6g5b<em1(IMPC)J>
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

>chr17:35114398-35114496 99bp CTTGTCCATTGCCGACCTT CTAGGAAGGGGAACACATCAG

Mut= 70 bp

Wt= 99 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

AGAGAGTGGCATCTCTGAAGGGGAGGCGGGGTATGCAGACAAATGAATTGATTTATTTTTTTTCTGCCCCCCCCTTTGTGTTTTTGTTTTTGTTTTTTTAATTTGTCTTTGAGAATTCATGGACTAAAGTCAGGTCTGCGTGAATGCCAGGAAGATAGAGGTTCGGGAACAGTGGGAAGCTATTAACCAGGGTTATAACTGTCCCCTCTCCTCTAGATACTACTGTTCGCTTCcacgtacggggctgtggacagcatcattcctaccgatgtcaagaaaggcatgtgatctaccaatcagactacttgtataaggctgattgctgccagtacgattactgcaactcctggtccagtgctcagcaccagagcaccctgcgtgggtccccaggaagccatctgggcatgcccctgtcagcgtctcagataaaacagttttaccaggccctgaacctctctctgcctcagcctggcttccatgctcataaggtgtctgagggcctggagtctctcattctgcccccggagctgggcttgtccattgccgaccttcgccaaatatacttgttcctcaacagttcaggacttctggtccttccCTGGGATAGACCCTGATGTGTTCCCCTTCCTAGATTCTGCTCTGTCTGAGAGACTCTTCCTTTGAGGGGCCCAGCATCCTGCTCTGCGGTCTGTGAACACTCAGATCCTCTGAGCCCTCTGATCTCTTAAGCTTTCTGCTCTACATGGCCAAATTCTGTATGGCCCCTGGATCTGGGGCTGGTTTGTCCTCCTCCTTTTCAGGAAACTTAAGGACGGTTCTCACCAAGGCTGACTTGACAAT

 

This mutation is a 365 bp deletion beginning at Chromosome 17 position 35,114,431 bp and ending after 35,114,795 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64375 CTT GTC CAT TGC CGA CCT T Wild type Forward A
64376 CTA GGA AGG GGA ACA CAT CAG Common A
64377 AAC TGT CCC CTC TCC TCT AGA TAC Mutant Forward A
64378 Fluorophore-1 CAG TTC AGG ACT TCT GGT CCT TC Quencher-1 WT Probe
64379 Fluorophore-2 ACT GTT CGC TTC CTG GGA TAG AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64375 0.40 uM
64376 0.40 uM
64377 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.