Stock No: 002250
Protocol 23475: Standard PCR Assay - Il10<tm1Cgn>
Version 2.3

Notes

Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands.
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 = 312 bp
Heterozygote = 137 bp and 312 bp
Wild type = 137 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR7376 CCA CAC GCG TCA CCT TAA TA Mutant Reverse A
oIMR9573 CTT GCA CTA CCA AAG CCA CA Common A
oIMR9574 GTT ATT GTC TTC CCG GCT GT 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
oIMR7376 0.50 uM
oIMR9573 0.50 uM
oIMR9574 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
004368 129(B6)-Il10tm1Cgn/J
002250 B10.129P2(B6)-Il10tm1Cgn/J
002251 B6.129P2-Il10tm1Cgn/J
005912 B6.Cg-Il10tm1Cgn (D3Mit49-D3Mit348)/Lt
004333 C.129P2(B6)-Il10tm1Cgn/J
003968 C3Bir.129P2(B6)-Il10tm1Cgn/LtJ
004291 NOD.129P2(Cg)-Il10tm1Cgn Il4tm1Cgn/DvsJ
004266 NOD.129P2(Cg)-Il10tm1Cgn/DvsJ
005346 NOD.Cg-Il10tm1Cgn Casp1tm1Sesh Casp4del/LtJ
004228 NOD.Cg-Il10tm1Cgn Il4tm1Cgn/Dvs
10 strains use this protocol