Protocol 36921: Probe Assay - Zfp541<em1(IMPC)J>Alternate1
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

>chr7:16076026+16076137 112bp CGAGTGCTTTTGGTTTGGA TGGATCAGGTTGAGCTTCTAGG

 

Mut= 96 bp

Wt= 112 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

AGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCTCTCTGTGTGTGTGTGTGAGAGAGAGACACACAGAGAGAGAGAGACAGAGAGAGAGCGAGTAAATAAAGCACTCACCTCCTCTAAGCTCTCTAATCAGATATAAGGTTGTTTCTAACATTTTACTAGAAAAAGCAGGTAGATTTTTTTTttTTATAAAAAGGTGAGAACCCAAGAGCAGAGCAAGATGGCGAGTGCTTTTGGTTTGGATAATGCATCTCTAGATAGACATTGTATGGGTAGGTTGGCTTctacccctgcttgtcttatcatactgctaccctagaagctcaacctgatccatacctgcacatcttgcaccaagacctggtgcagttctatagataaggcctggtccagacctcagctaagccttgtcgtctggccgtgcagagcctctgaggtgggggagtctcagactactgggacagggctctgtgctgagggtctatattgctttagcactcccctatcctatctcagcataacctgtgctttctccctttgtttagacctggggctgggcgcactcaaggtgcctaaagaggctgatgaaggaggcagggccactgggagtacaaggaaaggcaagcgacagcacagttcccctcagaatccactcctggactgcagcctgtgtgggaaagtgttcagcagcgccagttccctgagcaagcattacctgacgcacagccaggagagaaaacacgtgtgcaaagtctgcagcaaagccttcaagcggcaggaccacctgtatgtcaagaatctggccagaagggcagggggcatgcacctgtcctcctaccctctcccccatccatctggtcctctgttgagttcccacttctgtgcttctcagatgtatgtcccctgtcctccagtgtggactctgttctCTTCACACTGCTATTGTCTGGGGGCAGTCTCTTTACTAAGCCCCAAATTTACACCAGTGAGGAAATCCCTGTCTATTTGCAGACATGGCTCTTGTGGTTTCTAAATGTGATGAGTTACCAGGCGTGCTGGCACACTGGGGAGGTAGAAGCAGGAGGATCAAGAGTCTAAGGCCAGCCTCAACTACATACATTACAAGTTTAAAGCCCACC

This mutation is a 645 bp deletion beginning at Chromosome 7 position 16,076,086 bp and ending after 16,076,730 bp (GRCm38/mm10). In addition, there is an additional 2 bp deletion (TT) 98 bp before the 645 bp deletion site.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48990 GTA AAG AGA CTG CCC CCA GA Mutant Reverse A
48991 CGA GTG CTT TTG GTT TGG A Common A
50047 TGG ATC AGG TTG AGC TTC TAG G Wild type Reverse A
50048 Fluorophore-1 CCC TGC TTG TCT TAT CAT ACT GCT A Quencher-1 WT Probe
50049 Fluorophore-2 AGG TTG GCT TCT TCA CAC TGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48990 0.40 uM
48991 0.40 uM
50047 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.