Protocol 47748: Probe Assay - Tagln3<em1(IMPC)J>Alternate2
Version 3.0

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

>chr16:45722784-45722870 87bp ACCTCTTCTTTTCTCTGTCC TGAGAACACACTTCCCCT

Mutant=  99 bp

Wild Type = 87 bp

Sequence

Wt Sequence (deletion in lower case):

ATCCCACCTATTTGTTGGGCATCGTAGGGCTTGGCTTCTAAGAGTCTCTGAGAAGACCCAATTCAGTTCTTTGACGCATCACAAACTTAGATAGGATGAGTTAGTGAAAACAGCAGATCCCAGAGAGCAGATCAGAATCCTCCCGGTTCCCCGTGAGCCTCCAATGCATCCTCCAttaagaatgggagatcatgtcctcatcatccaggattttcaaggagggaactaggttctggctgcaagattgtggtcagctattgtctgtgaatgggctccctgggggtcttctgtggtgtgggtgtgtgtctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtagatgcaccactcacttctgacctgtgacatttattgcaaagggaaacacactaaccctctgtcccccacactccctttttaggtcctgtgcaagctgataaacagtttatatccaccagggcaagaacccatccccaagatctcagagtcaaagatggcttttaagcagatggagcaaatctctcagttcctgaaagctgccgaggtctatggtgtcaggaccactgacatttttcaaacagtggatctctgggaaggtaaactgccctcctggctttaggtctgtctcccctggactcttgtcccagtacctcttcttttctctgtcctctatttgcaaaagccacttacacagggattccctgtgctgcTTGGGGGAGGGGAAGTGTGTTCTCAGCTGTCATCCCAGGGACAGTGCTGGCCTTCCTGTTTGTGATGCTTTCTGGGTAGGGGTCCTGTCGTACAGAAGGACACATATTGAGGCTAGGGCAAGAACCTCAGCCAAGTAGTGGCTAAGTGTGATAGTGGAAGTGTCCACAGAGAAGAAGGGAACTGGCTACA

 

This mutation is a 545 bp deletion beginning at Chromosome 16 position 45,722,809 bp and ending after 45,723,353 bp (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
70858 ACC TCT TCT TTT CTC TGT CC Wild type Forward A
70859 TGA GAA CAC ACT TCC CCT Common A
70860 Fluorophore-1 TTT GCA AAA GCC ACT TAC AC Quencher-1 WT Probe
70861 TAG TGA AAA CAG CAG ATC CC Mutant Forward A
70862 Fluorophore-2 AGC CTC CAA TGC ATC CTC CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
70858 0.40 uM
70859 0.40 uM
70861 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.