Protocol 36528: Probe Assay - Qrsl1<em1(IMPC)J> Alt 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

>chr10:43604489-43604588 100bp GGATGTGAGCAGGCTGTGTA ATGGTTTGCCTCCTGTGCT

 

Mutant=  100 bp

Wild Type = 100 bp

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):

 CAGGAGGCAGAGGCAGGAGGGAGGCTTGAGTCTGCCAAAATGCAGTGTTTGTTGCTAAGTCTCTCTGGTCTTCTATACCATATGAATTTTAAGCCAAGTATAGATTACTCTATACAATTAGAGGAAAGTACACTTTT^c^acatggtagaattcaaatgcaattaaagaatgtcatttctcttaaatgaattctagggctctcggatcggacactggtgggtccaccaggaaccctgctgcccactgtgggattgttggtttcaaaccaagctatggcttggtttcacggcacggccttattcccctggtgaattcaatggacgtgccaggaatcctcaccagatgtgtggacgacacagcaattgtgttaggtatttctgcagttgcaaagcctatttgaaagcctcactgtaaaacattctgtctgtcacagtaagggtttctccttgcaggtacactggctggacatgaccccaaagattccaccacagtgagaaaccctgctcagccggcctcggttcccggcgggatggatgtgagcaggctgtgtataggaatcccaaaggtaactcgcttctgttgctttacaaaaagctgggaagttagatgcagagcacaggaggcaaaccatgaccagcctggtgggtttgaatctcttagccactacctctacaggccttgtgactgctgtcggccaagccagcccagtgtctccccttgagaaatggggatagaatccagatcctgagagttgagaggattaaatttgattatcctataaagcaaggctctgtagactttccttggaaagaaagcaacaccacgtTAGGGTTTGTAGATCGTAGATCTGGCCATTATAGTGAAAAAAGCAGCCATAGGCAGTTTGTTTATTTACATAAAGAAATATGTCTGTATACCTATAAAATTTTCTTTTCAAAAAGTGGATACACGGTAATATGTTGCCTGTGATCAGTGTGCTGATCCTTACTGTAAAGGACATAGGCATTCCTTCTTTAGTAAGGCCCC

This is a 687 bp deletion beginning at Chromosome 10 position 43,884,488 bp and ending after 43,885,174 bp (GRCm38/mm10). There is a single bp (C) insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49029 CTA AGT CTC TCT GGT CTT CTA TAC CAT Mutant Forward A
49030 CGA TCT ACA AAC CCT AGA AAA GTG Mutant Reverse A
49031 Fluorophore-1 TCC CAA AGG TAA CTC GCT TCT Quencher-1 WT Probe
49032 GGA TGT GAG CAG GCT GTG TA Wild type Forward A
49033 ATG GTT TGC CTC CTG TGC T Wild type Reverse A
49034 Fluorophore-2 AGC CAA GTA TAG ATT ACT CTA TAC AAT TAG AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49029 0.40 uM
49030 0.40 uM
49032 0.40 uM
49033 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.