Protocol 36521: Probe Assay - Lrif1<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

>chr3:106733028+106733143 116bp ACAGAATGCCCCCAGAAAAG CCTGTGATTCTTGGTCTCTGA

Mut= 124 bp

Wt= 116 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTGCCTCCTTTGGTTCAGCAAAAGATACTTGGAACTGCAACCACAAGTACCTCAGGAACAGTTGAGGCCTCCCAAATACCAACAGTTGTTTATGTACATCCTGTAAATTCAGTGAAATTTGTTGTTACCAAGAAAACTCAGACTATTTACCCCAAACCAGTGACATTCAATACCTTGCAAATTCCACCAAATGTTGCTACAGAAACACAATTAAAAGGTGGTCAGCATCCTCAAGCTGCTCCAGTGAACTCGatttttcaggagtatctacagcctggaatcccatgtattattcctgttaagtcttcaaataatgtggctacaaaggttttaaacacttttgtaggtaggaaaaatctgggagataatacaatagatacacctctgttgaataccaacagttctggcaggacacactccgtaagtgaacctattaaagataatgctttgataatgtttaatgggaaagtctggttgaatgaaaaggggacatgcggtctgccatcaaaaattgaccagcagaattctgtttcttctgatattccactaaaagattcatcacagctagtaagttcaagtatagtcacagaaatatccagagagattttaaatagtgttttggttaaaagtaaatcttttcagttgaagacaaaatcactttcaaattcccagcttgcttccatggccaatctaagagcagagaagaatgaaaaagtggagagaccatctttttctgtcacaaatccacatactatgaaccagtccactcactgccttaaacagagtaagactgtatttataaacccagtttttccagatggatttaggacaggacagaatgcccccagaaaaggaaatctcgtccagaacatagagaaaatatgttcctctgttgATGCAGCAACTGTTACTTCTCAACAGTGTGTTTTCAGAGACCAAGAATCACAGGTAATTTTCCCACATCAGGATGCTCTTTATATATATATATATATGTGGAAACTATTTCTTAATTCTCAAAATTATCTGATACCTGTCTCCTACACACACACCCCATCACAAGGCTTGCTTAAAATTTTTATCTGAATAAATATTTGTGTACAAGTAGTAGAGCCTAAGCAATATCTTTCTCTTTCAT

This mutation is a 643 bp deletion beginning at Chromosome 3 position 106,732,447 bp and ending after 106,733,089 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49000 ACA GAA TGC CCC CAG AAA AG Wild type Forward A
49001 CCT GTG ATT CTT GGT CTC TGA Common A
49002 TCC ACC AAA TGT TGC TAC AGA Mutant Forward A
49003 Fluorophore-1 CCA GAA CAT AGA GAA AAT ATG TTC CTC Quencher-1 WT Probe
49004 Fluorophore-2 CCA GTG AAC TCG ATG CAG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49000 0.40 uM
49001 0.40 uM
49002 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.