Protocol 36269: Probe Assay - Zgrf1<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

>chr3:127567556+127567655 100bp AGATGGAAAGGCATTTTTGC GAAACTTCTATGGCAGGTGATTTC

Mut= 122 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TAGTATGCGAAAGCATATCCCTATTAAGATCAGTAAGAGAACTGGAGGGAGGGCTCAGCAGCTAAGAATTTCCTGCTCGTGCTGAGGAACCTGATTTCGGTAAAGCATCCCCTTCTGGTCTCTGTGGCACCCGACATGAATATAGTGCATATATGCATGTGTAGACAGATATGCGTGCCAAATAAAGAAATCTGTCTGTCTAGTGTATCTATTTGAGACAGGGCTTCTCCATGTAGCCTTGGCTGTCCTGAAACTTACTGTAAACTAGGTTGGCTTTGAATCTACAATCTATCTACCTCCACTTTCTGAGTACTGGGATTAAAGGCATGTACCCACATATGGTCATATTTTTTTTTTAAAAGTAATCAGTAATGTTTTCTAAATATATTGAATTTTTGCCATAAGAATGTACCAgcctatacagaatggtatactcatactctgtatagtatactatacggaatctttactcatactccagattaaatgattatcaagatattactttttaaagtgtattgcttcagttttagatggaaaggcatttttgcaaaataccgtgatgcatatgacaaatgaggctgtttgattctttcattgcagccaggaaatcacctgccatagaagtttcgcgtgctctgctagagtgtccttgtgagcatcgggcttctcagcaggtacgtatgtatctctctctaactactatgtttttcttgtgtagaaaatagtgacagattcatatgcatgtagcatatactttgattatactttctcactactctcccctgcccccattctggtctgtaTTCGTTTAAGCATCATTCGTGTCATACAGTTGATAAGAATTGTCTTGTTAGGGCTCCTGTTGCTATGATTGTTACTATGATGAAACACCATCACCAAAAGCAGCTTGGGGATCCGAGTGGTTATCTCAGCTTACAGTTCCAGGCAACAGTCCATTACTGGGGAAGTCAAGGCAGGAACTCAAGTCAGGCAATAGGGCAGGAATCTAGAGGCAGGAGATGGTGCAGAGACCATGGAGGGAGGAGAGCTGCTTACTGGCTTGCTTGATCTGCTTTCTTATAGAGCCCAGGACTATCAGACCAGGGACATGCCACCCACAATCCTCCCCCATCAATCACTAATTAAGAAATTGCCTTTCAGCCACATCTTTTTTTTCTTTCCTTTGACTTCCACATCATAGTCAATCATTGAAGGAAGTCAGGAACCTGGAGGCTGGAGCTGATA

This mutation is a 392 bp deletion beginning at Chromosome 3 position 127,567,439 bp and ending after 127,567,830 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48243 AGA TGG AAA GGC ATT TTT GC Wild type Forward A
48244 GAA ACT TCT ATG GCA GGT GAT TTC Wild type Reverse A
48245 GGC ATG TAC CCA CAT ATG GTC Mutant Forward A
48246 AAC TGT ATG ACA CGA ATG ATG C Mutant Reverse A
48247 Fluorophore-1 ATA TGA CAA ATG AGG CTG TTT GAT T Quencher-1 WT Probe
48248 Fluorophore-2 TTG CCA TAA GAA TGT ACC ATT CG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48243 0.40 uM
48244 0.40 uM
48245 0.40 uM
48246 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.