Stock No: 000469
Protocol 17673: End Point Analysis Assay - H2-T18<a/b>-EP-Alternate 2
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

H2-T18<a> = C (Jrs 469, 4804, 465) = "hom"

H2-T18<b>= T (Jrs 666, 664, 457, 438) = "wt"

 

 

Sequence

Tctgatgagttattgatctagctatgataa(T/C)cagtgattg(ATTG/----)ctattat(A/C)acagtcaca

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25550 Fluorophore-1 AAT CAG TGA TTG ATT GCT ATT ATA AC Quencher-1 WT Probe A
25551 Fluorophore-2 TAA CCA GTG ATT GCT ATT ATC ACA G Quencher-2 MUT Probe A
25833 AGT CCA AAG TTT TCA CAT TAA TCA Forward A
25834 TCA ATG AGA AGC CCT TTT AAG TC Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
25550 0.40 uM
25551 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 4.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
000469 B10.A-H2a H2-T18a/SgSnJ
000465 B10.BR-H2k2 H2-T18a/SgSnJ
004804 B10.BR-H2k2 H2-T18a/SgSnJJrep
036602 B10.Cg-H2k2 H2-T18a Tg(SV40-HA)HA104Ajca/VradMmjax
4 strains use this protocol