Protocol 36024: Probe Assay - St13<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

>chr15:81388589-81388683 95bp TGCCATCTCTCCAGCTTACC AAGCTAGTTTCACCTAAACCCTGA

Mutant=  111 bp

Wild Type = 95 bp

<p>TGS&nbsp;Design, DJL 6-6-19</p> <p>&nbsp;</p> <pre> <tt>&gt;<a href="http://genome.ucsc.edu/cgi-bin/hgTracks?hgsid=729122631_udwo4AzzARLb9ZLIQ14NguIs7JSJ&amp;db=mm10&amp;position=chr15:81388589-81388683&amp;hgPcrResult=pack">chr15:81388589-81388683</a> 95bp TGCCATCTCTCCAGCTTACC AAGCTAGTTTCACCTAAACCCTGA</tt></pre> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;Validator:&nbsp;&nbsp; DSW&nbsp; Date:&nbsp;&nbsp; 8/30/19 &nbsp;&nbsp;&nbsp; Req:&nbsp;&nbsp;&nbsp; 573002</p> <p>Charge Assay Fee: Yes - Import</p> <p>Mutant reverse has low GC&nbsp;content, not a great region.</p> <p>Assay reveiwed, potentially hom lethal - need more samples to confirm. DSK 12/30/19</p> <p>&nbsp;</p>

Sequence

Wt Sequence (deletions in lower case):

ATGTATGTGGTACTTTCAGAAGCCAGATCCCCTGGTACTGGAGTTGCCATCTCTCCAGCTTACCTAGTAATAGCTAGTAGTAGTTATTTctagcagggaagtagttaataatgtatcagggtttaggtgaaactagcttatagttctactagcaagtagttcattttgaggaagggatatacctttaaccttcatcctattggatagtacttagccctgagctccacacaaatcagttcatctcattttacagtttcaagccagtggctgttaactgtaccacactcgtaatttaggtttcaatctcatggttttcaccttttgcagAAATTGACAATGAAGGTGTAATTGAACCAGACACTGATGCCCCTCAGGAAATGGGAGATGAAAATGCAGAGgtaagtaagtgacattttaggaaaagtgaagttccccggggttggattattttataatataactcccaagatgtgtatgataaatcttcctttgactgatgacatactttataatttCACTAAAAGATTCAAAAATTCCCATGTTGTTTTTAACTATTATCTCTGACGTCTTGTATTTTGTTTTACTTGTTCTATCAATTTACTTCATTTTGACTAACCTGTAAAATTATTTCTCTAAAATAGATACCTACAATTAGAATTGTTCTTTTTTTTTTTTTTTTGAGATGATTTTGTCTTTTTTTCAAGAC

 

This is a 426 bp deletion beginning at Chromosome 15 position 81,388,213 bp and ending after 81,388,638 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47603 TGC CAT CTC TCC AGC TTA CC Common A
47604 AAG CTA GTT TCA CCT AAA CCC TGA Wild type Reverse A
47605 AAA CAA AAT ACA AGA CGT CAG AGA Mutant Reverse A
47606 Fluorophore-1 TAT TTC TAG CAG GGA AGT AGT TAA TAA TG Quencher-1 WT Probe
47607 Fluorophore-2 AGC TAG TAG TAG TTA TTT CAC TAA AAG ATT CAA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47603 0.40 uM
47604 0.40 uM
47605 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.