Protocol 36045: Probe Assay - Luc7l3<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

>chr11:94303412-94303531 120bp AGTAACTAGCTCCAACCCAGTG GGAGTCTCTTCTACCCATGCTC

Mut= 131 bp

Wt= 120 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCAGCTATTTGAGAAACTGAGACAGTAAGATCTACGTTCAAGGCCAGTCTGTGCACTTAACGGGGAGTTTTATCACAAAACAAGGGGCTGGAAAATACAGCTCAGTGCTAAAGCACTTTGCTAACTTGTGTTCAATATATAGTACCACCAAAATAAACAGCCAGCTGATTATTAACCAACTGCAATACAGTATATAGTGCCTTTCCCtttgataggataacatctaaaaatatacttcatgtaagtttaggctttgagacggggtgatagggaatatatatatatggcttgtaatgtttaaaagtttatttgttttcactttatcaggtatgagaagagttctcgctttatgaaagttggctatgagagagattttttgcgatacttacagagtttacttgccgaggtagagcgcagaattagacgaggtcatgctcgtttggcattatctcaaaatcagcagtcttctggggtgagtatgaaatggacttagagtttgtaaagaactctgttctctgtagatgtggctgtctcatatcattttaaactcgacttccattcattactctgtcctttagaatacataaggtttgaggagcaccttccttctgttgtttgagggttagggaagcgtctccccccttccgcgtgcatgtttggtctgatggtgctgctggaggtcatgggctttgtatatgctagcacgtgctccaccactaaaccacacgttcgttctccagcctctgctccatgctaggaagaccagggcgggtggtcatcctagtaactagctccaacccagtgacttacttcacctccaccatgagaataggataagcaaaggggccaagactgtgtaaggagatTGCTTTTCTGAGGAGAGCATGGGTAGAAGAGACTCCTTTCTGTGACCTTTGAAGGCAAGCAAGGAGCTAGCTAGCCAGCAGAATCAAACGGAAGCCGCTCGGGTTGGGCTGCAGTAAAGCATAAAGACAGAAAAGGAGAGGAGGTTATTTCCCCAGACAGCTGTGACAAAGCTAGAGTGCAGGTGTAAAGATCTTAAGTATATTTCT

This mutation is a 659 bp deletion beginning at Chromosome 11 position 94,303,448 bp and ending after 94,304,106 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47683 AGT AAC TAG CTC CAA CCC AGT G Wild type Forward A
47684 GGA GTC TCT TCT ACC CAT GCT C Common A
47685 GCA CTT TGC TAA CTT GTG TTC A Mutant Forward A
47686 Fluorophore-1 AAG CAA AGG GGC CAA GAC Quencher-1 WT Probe
47687 Fluorophore-2 ACA GTA TAT AGT GCC TTT CCC TGC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47683 0.40 uM
47684 0.40 uM
47685 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.