Protocol 33401: Probe Assay - Ralgps1<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

>chr2:33260679-33260761 83bp GGAGATTGGGCCCCTTTAG TGGCAAAAGGAAAGACCTGA

Mut= 85 bp

Wt= 83 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TTCTCGTGGCACGTCCGCCTGAGTCGCATGGCAATTGATAGCATATTTCAGCATGAGGCACTGGGGAGAAGAGACTGGCTAGGGCTTTGGAGTGACGCTCCTGGGGACactctcTGAGCCCTGTGCTGTATTTgccctcacagagatctatgttgggtgtgaaatgaaggagctagtgagcatcccgtggccagtggacgaggtggctctgaggctgactcctgggagattgggcccctttagcacctgtacttctatccagggtttgatagGTTGTGGgcatacattcaggtctttccttttgccatttgtcatagcccctggcctgtcagcttttctgattgattttttcccctccagataaatttcttttcattccagcttttgaatcgaaaagacaagactacttttgagaaacttgactacctgatgtccaaagaagacaactacaagcgaacccgggactacatccgaagtctgaagatggtgcccagcattccctatctaggtatgagtctcagttattttattattaactttccaggcaacataacatgctcagggccaaataagatgtttttacagtttctagttgagatgtttccaggaagcagcagggatcatggataaagaggtggtttggttggctccttggttggagaagGCTGTTGATGGTTCCCAGGATAGGAACACTAGTGaggtgGAAACAAGGCAACTCTGTCTTATGTACCTGGACTCTGAGGGCCCGGGCTACCGAGCTGTGAGGGCTCCAGGCATCCAGGCTGCAGCCCAGATAGGTCATGATGTTGGTAGGTCATGCTGTCT

This mutation is a 524 bp deletion beginning at Chromosome 2 position 33,260,322 bp for 139 bp then a 7 bp (GTTGTGG) endogenous retention followed by an additional 385 bp deletion ending after 33,260,852 bp (GRCm38/mm10). In addition, there is a 6 bp deletion (ACTCTC) 19 bp before the 524 bp deletion as well as a 5 bp deletion (GTGGAG) 31 bp after the 524 bp deletion, neither of which are expected to alter the results of the exon deletion. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41338 GGA GAT TGG GCC CCT TTA G Wild type Forward A
41339 TGG CAA AAG GAA AGA CCT GA Wild type Reverse A
41340 ACT GGC TAG GGC TTT GGA GT Mutant Forward A
41341 CCT ATC CTG GGA ACC ATC AA Mutant Reverse A
41342 Fluorophore-1 TTT GAT AGG TTG TGG GCA TAC A Quencher-1 WT Probe
41343 Fluorophore-2 TGC TGT ATT TGT TGT GGG CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41338 0.40 uM
41339 0.40 uM
41340 0.40 uM
41341 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.