Protocol 28118: Separated PCR Assay - Tlr4<lps-del>
Version 4.2

Notes

Melting curve analysis is done using a Roche Light Cycler 480.
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

Het= 390 bp and 140 bp
MUT = 140 bp
WT = 390 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8365 GCA AGT TTC TAT ATG CAT TCT C Mutant Forward A
oIMR8366 CCT CCA TTT CCA ATA GGT AG Mutant Reverse A
oIMR8367 ATA TGC ATG ATC AAC ACC ACA G Wild type Forward A
oIMR8368 TTT CCA TTG CTG CCC TAT AG Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR8365 0.50 uM
oIMR8366 0.50 uM
oIMR8367 0.50 uM
oIMR8368 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
007227 B6.B10ScN-Tlr4lps-del/JthJ
005315 B6;C3H-Tg(Scgb1a1-Scnn1b)6608Bouc/J
003752 C57BL/10ScNJ
033704 NOD.Cg-Tlr4lps-del Prkdcscid Il2rgtm1Wjl/SzJ
4 strains use this protocol