Protocol 36345: Probe Assay - Snx6<em1(IMPC)J>-alternate4
Version 2.0

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=  103 bp

Wild Type = 87 bp 

>chr12:54770717-54770803 87bp CACGGGAAAAACTGCAGAAG ACGCTTCCAGTTCCTGTTTC

Sequence

Wt Sequence (deletion in lowercase):
CTGAGTTCGAGGCCAGCCTGGTCTACAAAGTGAGTTCCAGGACAGCCAGGGCTATACAGAGAAACCCTGTCTAGAAAAACAAACAAACAAATAAACAAAAAAAGAAATCGTTCGTTAAAGTtagttatttggttttgttatagatcccaccagcaccaccaagacctgattttgatgcttcacgggaaaaactgcagaagcttggagaaggggaagggtcgatgacaaaggaagaattcacaaagatgaaacaggaactggaagcgtatgtgatgtttttttaacaattacttttgttaattgtgctctctgatactgtagtgactgaaaaagccagggtaagtcataagagtcactatcagagcctcttcaggaaaagagcaccattgtcttcattgtcagatttaaaattatttaaatagactctgaaaattgaaaattgaggatctaaaaccttattgaagtactaggaaggcaatatgaccttaaaacggtatccatttataaggcctCAGTCATCCTCACCTGTCTCTGTTTTCTCAGCGTGTATCTGCCCTACAGCTTCCATTTCTTGTAGAAAGTGTGCTGAGCATCTCTTTACATTTTTTCTTTTATGGCCTGGTCTATAGAGTGAGTTCCAGGACAGCCAA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47972 TAG GGC AGA TAC ACG CTG AG Common A
48127 CAC GGG AAA AAC TGC AGA AG Wild type Forward A
48128 ACG CTT CCA GTT CCT GTT TC Wild type Reverse A
48129 Fluorophore-1 TTG GAG AAG GGG AAG GGT C Quencher-1 WT Probe
48130 CCC TGT CTA GAA AAA CAA ACA AAC Mutant Forward A
48131 Fluorophore-2 CGT TCG TTA AAG TCA GTC ATC CTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47972 0.40 uM
48127 0.40 uM
48128 0.40 uM
48130 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.