Protocol 33345: Probe Assay - Ralgapa1<em1(IMPC)J>-Alternative 1
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

>chr12:55795118-55795263 146bp CTCCAATTCTCAGACCTAGAACCT CAACCACTGGCTATTGTTTTCT

Mut= 125 bp

Wt= 146 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 

AGTGTAAATTAAGTTTTCTTTTTATCCTGGGGATTGAATCCAAGGCCTAGTACATGCTAAGTGTGGGTTTTCTCATTAAGCTACTCCAATTCTCAGACCTAGAACCTGAATTTAGAATAATTATGCTAAGGAGTAGtgagAAGGTTTATAGTATTgtctatataataaatagaccatgatttagcatttgtctaaaagaatcattgaagaaaacaatagccagtggttgaaatatttaagttcattaagtagttgttaatagtgagtgtatttaaagtatttttctgttccaaatatttcaggtcacaagtctcaaagagaggaattagatgctatactttttatttttgaggtaagtgctatgaatcatactgtttaaactttttcaaaaaattattctgagaatataaagaaatatattaaaattattagacatatagtgaaacatagttaacctgccagccttttGAAGAACTTAATTAACTTTAAAAATGTATCTGGCTGAACTTGTGTATTTTTGAAGTATAATGTATTTTTTAGCAACAAAATTATGCATCTGACAATATAAAATTCATTTGATAGCATTTTTATAAAGTGAAAAAAAATTTTTTAA

This mutation is a 313 bp deletion beginning at Chromosome 12 position 55,794,879 bp and ending after 55,795,191 bp (GRCm38/mm10). In addition there is a 4 bp deletion (TGAG) 15 bp before the 313 bp deletion that will not alter the results of the mutation.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39256 CTC CAA TTC TCA GAC CTA GAA CCT Common A
39257 CAA CCA CTG GCT ATT GTT TTC T Wild type Reverse A
39258 TCA AAA ATA CAC AAG TTC AGC CAG A Mutant Reverse A
39259 Fluorophore-1 AGA CCA TGA TTT AGC ATT TGT CTA AAA Quencher-1 WT Probe
41194 Fluorophore-2 AGG AGT AGA AGG TTT ATA GTA TTG AAG AAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39256 0.40 uM
39257 0.40 uM
39258 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.