Protocol 35544: Probe Assay - Slc6a16<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

>chr7:45261370+45261473 104bp GCTACAAGCCCTGTTCCTTC CTCGGTTAGCACGTCAGTTCT

Mutant=  110 bp

Wild Type = 104 bp

Sequence

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

TGACGTCTCTGAGATGTGAGAGCAGGATAGAAAGCAGGAGTCCGAGCACCCAGGGTCTGGGAAAGTGGAGGTCAGGGTTTTGAGCATAACTCGGTGGGTTGGCCAGGCAGGATGTTGGTGGGGAAGGGCAATGACCAGGCGTCTGAGACTCCTAGAAGTAAGAAGGTTGGCCAGTGacggatgcggggcagtcaagccttaaccttgtggcctgagtgagggtgctgctggtgagcagtcttcctagttgcataaagaaggacagacatgagccgaggctctcggaacccttctgccttctctccacaggcagcttctttatagcgtacatcttgatgctgttgggcattgggattccacttctcttcctggagatggcagttggtcagagggcacagcagagcagtgcggacatgtggaagaacctgagcccctggtttggtggagtggggtacagcatggttatggtgaggagccccaggcttcacaaacctgcctgccccacagtgctcctctccctcccctctggagaaaatcccacggctgccctggtgtggaccatgctccttcagcccctccaaactttgtcagtcccttctctctccctgtccctggtcttcctccccagtcatgtctccactggcctgcaggtgtgcttcatcaccaacacctacctgaacgtgttcaattcttggatcctcttctacatgagccacatattctactttgttgtcccatgggaccagtgtcccttacaaaggaattccagtaactttggtgaggaaggagacgtgggagaatgaagggacttgggaagaggaggagagaggagggaggggcacgaagagaaagacaggagaggagggaggcagggggttggggcttgccttatctagcaggcctgtccaaagccatgttcccccagatcctgaatgtgaacaggcaacatcctacacgtacttttggtaccggaagaccttgaaagcctcagacagaattgaggatggagggcaaccgtccttcagcctgggcatgtctctctttctgtccttctgtctcatttgtgctttcttggtcaatgggatcaagtccattgggaaggtgagctgcacttggcccttggcattctcaagtgctacgaccttgttttctccttcctggccagttatccccaaaagctttttttccgagtcctcgtccagcccccttagctctacaaagctgagtcgcttgacaagcagctgatgtgctgccctccccttgtaggtcctgtttgtcttgctactggttccctattccatcatcgtctgcttcctcatccggactctgtcgatggacggcgcagaatacgggcttaagcacctgctgatcctcaaggtgaggttcttcctgggttccctgagctctgggaccagcacggctgcctgctgttgtctcccgtacctcctcctttcccttccctctcatagttgttctctcccaggtcctgatgtccctattgtacctgtcttctcctttttaggtggcgagcatctctgacttaactatttggtgccatgcaggtatccaggtcttgtttgatatcggcctgggctttggccccattgtctccttagcctcacacgtacccgacttcaacaactgtatggccgatgccttcttaatggctctgttcaagataatcaccttactgatgaccacacccttcctcctctccatcctgggcttctgggccactacaactacacatcactgctgtaagaagtaaggccagtccctggcaggtcacaacccattcacacaccttacccgcagggaggctacaagccctgttccttcttatctgagagcagagccccttcctaacattagaaagctccagAGGTCGTCAGGCCGCACCTCAGAACTGACGTGCTAACCGAGGCCAAGAGTGGTAGGGAGGCAAAGGTCACCCCGGCACAGAGA

 

This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences GTTGGCCAGTGACGGATGCG and CTAACATTAGAAAGCTCCAG, which resulted in a 1706 bp deletion beginning at Chromosome 7 position 45,259,727 bp and ending after 45,261,432 bp (GRCm38/mm10). This mutation deletes ENSMUSE00001034222 and ENSMUSE00000967546 (exons 4 and 8) and 892 bp of flanking intronic sequence including the splice acceptor and donor and is predicted to cause an early truncation after amino acids 160.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46343 GCT ACA AGC CCT GTT CCT TC Wild type Forward A
46344 CTC GGT TAG CAC GTC AGT TCT Common A
46345 CAG GAT GTT GGT GGG GAA G Mutant Forward A
46347 Fluorophore-1 AGA GCA GAG CCC CTT CCT AA Quencher-1 WT Probe
46350 Fluorophore-2 TTG GCC AGT GAG GTC GTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46343 0.40 uM
46344 0.40 uM
46345 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.