Protocol 35511: End Point Analysis Assay - Champ1<em2Lutzy>-EP
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

Mutant = T

Wild type = CTC 

>chr8:13878194+13878265 72bp AGCCTTTGACCCAGCAGAG AAGCTCGGGCTCGTTTTCT

Sequence

WT sequence (with mutation in parentheses):
GAAAGAGCAGCCGAGCAAAGACACCGAGTCTGGGAAAAGCCCTTCCCCTCCTGAGCGGCAGAACCCAGCCTTTGACCCAGCAGAGGCGAGGCCCACTCCAGCC(ctc/t)CCCATGGAAGCACAGAAAACGAGCCCGAGCTTGTGTCCAGAGTCACAGGCTTCTGGTCCTCCAGTTCTGGAGCCTCAGG
 
Mutation is del3 (ctc) and ins1 (t)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46254 AGC CTT TGA CCC AGC AGA G Forward A
46255 AAG CTC GGG CTC GTT TTC T Reverse A
46256 Fluorophore-1 CCA GCC CTC CCC ATG G Quencher-1 WT Probe
46257 Fluorophore-2 CTC CAG CCT CCC ATG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46254 0.40 uM
46255 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.