Stock No: 001060
Protocol 40429: End Point Analysis Assay - H2-K<bm1>End Point Alternate1
Version 5.0

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

Sequence

Wild type:

CCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACA
AGTGGGAGCAGGCTGGTGaaGCAGAGagactCAGGGCCTACCTGGAGGGCACGTGCGTGGAGT
GGCTCCGCAGATACCTGAAGAACGGGAACG
 

Mutant:

CCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACA
AGTGGGAGCAGGCTGGTGctGCAGAGtattaCAGGGCCTACCTGGAGGGCACGTGCGTGGAGT
GGCTCCGCAGATACCTGAAGAACGGGAACG

 

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
15058 CCT GAA CGA AGA CCT GAA AAC Forward A
15059 CGT TCC CGT TCT TCA GGT ATC Reverse A
56016 Fluorophore-1 TGG TGA AGC AGA GAG ACT CAG GG Quencher-1 WT Probe
56017 Fluorophore-2 CTG GTG CTG CAG AGT ATT ACA GGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
15058 0.40 uM
15059 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.