Protocol 39584: Probe Assay - Tg(Tcra,Tcrb)9Rest-ChrY
Version 1.0

Notes

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

Tg=  117 bp

IPC = 74 bp

Transgene insertion site is known to be on mouse Chr Y.

This assay is NOT able to be used for copy number evaluation.  If this is required, it is suggested to type by qPCR.

Sequence

Tg Sequence:

TATGGCAGCAGAATCTTTATGCATAAATAGAGGCAGATAACTAAATGCAGGATAATGACTTGCTCTAGTGTGTATACATGGTTTTCAGGACTCTCCTTAAACAGCCTCTTCTTGTGTGCTACATTATCCTTAGCCATCACTAGGATTTTCTTTTTCTCATTTGCTGTTTCCAAGACTTTAAAGACGCTGAGATTTCTGTGTTTCTATTCAAACAATCCTCTCATTTGGAACTTAATTGTATTAAATCTAGCTAAAAATCGTTTCTCCTTTTAATGTACACACACACACACACACACACACACACACACACACACACAGTGTTTCTCCTTGAAGACAGACCCTGAGATTGCACAGAGTTCCAACAGGAATGTTCTAGGATGAATTCAGAGCAGACAGCACATGAGAAAATTGGCATCGGTTTGCCACATAAGCATTGTCATGTTTGTGACATATTTTCTGTTTAAATATTTTTAAATGAGACAATTTGGAGGAGCTACAGTCAAAGTACTTTGTAAGCTTCCAGGGAGGAAAAGGACACCTGCCAAAAGTTAGACTTTGTAATAGTACTTGCAAAATATGATCTCCTTCAATATCTAACAATGCATACAGATAACATTTGCTGGTGAAAAAAAAATGAAATCTAGAAAAACTACATAACTGCCCTCAGGTCTTAGAAACTAGGAAATCTAGACTTGAAGTCAAATGTTTTACTTGTAGCTTTGCCTGGTACAAATCAATGGAAAACATATTCTCACAAAAATATACTTACATATTTTTACTATATTTGTTTGTAATCGTTGATAGCTAAAAGCGGGTGTTTTATCTGGGAAATGACCAAAGCACTGGTGTGTCTAGTGCATAGATTATTTCTCAGCAATTAAAAAACAATGAGTACTCCCACCTTATCCTCTCTTTCTCCATTTATTGTTCTCCCTCTGATACACAGACGCAAAATTTTCAATTAACAGGAACTCCTCTGTAAGTATCAGGAAAGTGCTACACACTACAAAACAAGCATTGGGAAATTTCCAGGACAATACTCTCATTTTAATTTTGTAGGAAAAAAAAAACTCCCTTAGATACCTTAAAGAGGCAGAGACTCACCCCTTCTAAACATAGGTAGAATAATAGGTGACTGTTGATAAATATTCTGGAGCAAAGCAGCAAGTGAGAAATAGGAAGAGAAGGAAGAGGAGAAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGTACTCAAAAGAAAGAAACCTTTAAAAGCCAGTGTTCATTAAGAATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTATCTTTTTTGATTTATTTAATCTTTTCTTCATACTCCAGGTGTTATACCCCTCTAGGTCCCCCCTCTGATTGTTCCACATTCCATACCACCACCCACCAATTCCACAAAATTGTCTTCAACCCCCACCTCACCAGAACTACCCAAGAGACTGTCTGTGGCCTCCAGTCTCTTGAGGGTTTGGTGCATCTTCTCTGACTAAACCCA

 

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
54159 CAA ATG TTT TAC TTG TAG CTT TGC Transgene Forward A
54160 ACC CGC TTT TAG CTA TCA ACG AT Transgene Reverse A
54161 Fluorophore-1 CTG GTA CAA ATC AAT GGA AAA CAT Quencher-1 Tg Probe
oIMR1544 CAC GTG GGC TCC AGC ATT Internal Positive Control Forward A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
54159 0.40 uM
54160 0.40 uM
oIMR1544 0.40 uM
oIMR3580 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.