Protocol 31827: Probe Assay - Spata3<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

Mutant=  136 bp

Wild Type = 139 bp

>chr1:86024644+86024782 139bp AACAATGTCTCTGGTTTGGTG GTGAATGGCTAACCAATGTGT

Sequence

Sequence (deletion in lower case, insertion in carrots)

GTAGAAAGCAAAACCTTCCAGCACCACATCTTTAAACCTTTAAAACGATAGGGTATTGAAGAGGCAAAGGAGGGACTGTCCATGGCCTACTGAATTTGTTAACTTTCTCATCACCGTGACCCAGTTATCTGAGAAATTACTGAAACGATGACTTATTCACGTTGGCTCACGGTTTTAAGAGTATCCACGTATCACGGCAAGGTGTAGCTCACAGTGAATCTGCTAGGAAGTGGAGAGAGGGATTATGTGAAGGGCCCAAGGCAAGATATAACCTGCAAAGATGTCCCACTGATGCCCTACAacacctgctagaatccgcctcttagcctttaccaactcccaataacatgacttctagaactgtcctagagactgtgtcccactcccaagaaccaggtgcagtcctcatctctctccttctctgtccagcagtgcctctgacctatgtggccccacgctcctgctcctgtgcagcctgcccgggcagctccgcttgctggcatcgtctggggctttgccatagccgaatctttgatgtcctcctacctcgggactggtcatccatgccagggagaggagtcccaaaccttctcaccttctacaggttccgcatgaacgggagggctaaaaagccttgggtgtagagttcccagggccaggctcggtcctcagatctggttggcaactgtgtgtaacttcctccaaacatcataggcttttcaaaagcaaagccgttccttctaaataacaatgtctctggtttggtgggccagctctttgtctgttcctttttctatgaaaccgtcttctctgctgacagcaccctcccaccCAGCCCATTGTAAACTCCAGAGCTGTGTGATTACACATTGGTTAGCCATTCACTTTTTTTTTTTTGACCCATACCATACACAGGGAGtgtctattta^GGTTTTTT^TTAAACGGCATGTTAATTATCCCTGGAACTCAATGATAAACCTGATCGCGTAACTCATAGGTCCCTTCTGCCAATAATAGCAAAATCTGTCCCACACAGAACATGCCATGCTTTTACCAGGAGTCCTCACAGGGGCCATGTTTTCTAGACCAGTCTATTAG

531 bp deletion beginning at Chromosome 1 positive strand position 86024199 bp and ending after 86024729 bp (GRCm38/mm10). In addition there is an indel (8bp insertion GGTTTTTT and a 10 bp deletion TGTCTATTTA) 85 bp after the 531 bp deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37769 TCT GCT AGG AAG TGG AGA GAG G Mutant Forward A
37771 GTG AAT GGC TAA CCA ATG TGT Common A
37772 AAC AAT GTC TCT GGT TTG GTG Wild type Forward A
37773 Fluorophore-1 AAA GAT GTC CCA CTG ATG CC Quencher-1 MUT Probe
37774 Fluorophore-2 CTG CTG ACA GCA CCC TCC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37769 0.40 uM
37771 0.40 uM
37772 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.