Protocol 22825: Standard PCR Assay - B2m<tm1Unc>

NOD.Cg-B2m^tm1Unc Mcph1^{Tg(HLA-A2.1)1Enge} Tg(B2M)55Hpl/DvsJ

Stock No: 004548

Version 2.3

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 = ~410 bp
Heterozygote = ~410 bp and 261 bp
Wild type = 261 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
oIMR0160		TCT GGA CGA AGA GCA TCA GGG		Mutant Forward	A
oIMR0184		TAT CAG TCT CAG TGG GGG TG		Common	A
oIMR0185		CTG AGC TCT GTT TTC GTC TG		Wild type Forward	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
oIMR0160	0.50 uM
oIMR0184	0.50 uM
oIMR0185	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
002454	B10.129P2(B6)-B2m^tm1Unc/J
003533	B6.129P-B2m^tm1Unc-rs^2J/J
002087	B6.129P2-B2m^tm1Unc/DcrJ
002070	B6;129P2-B2m^tm1Unc/J
002449	BXSB.129P2(B6)-B2m^tm1Unc/J
002420	C.129P2(B6)-B2m^tm1Unc/J
004040	C3.129P2(B6)-B2m^tm1Unc/Dcr
002439	C3.129P2(B6)-B2m^tm1Unc/J
002453	MRL.129P2(B6)-B2m^tm1Unc/J
002455	MRL.Cg-B2m^tm1Unc Fas^lpr
005356	NOD.129(B6)-B2m^tm1Unc Ciita^tm1Ccum/BhsJ
010565	NOD.129P2(B6)-B2m^tm1Unc Tg(HLA-A24/H2-D/B2M)3Dvs/J
002309	NOD.129P2(B6)-B2m^tm1Unc/J
008542	NOD.Cg-B2m^tm1Unc Tg(GFAP-B2m)9Mdos/J
006611	NOD.Cg-B2m^tm1Unc Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ
004548	NOD.Cg-B2m^tm1Unc Mcph1^Tg(HLA-A2.1)1Enge Tg(B2M)55Hpl/DvsJ
010636	NOD.Cg-B2m^tm1Unc Prkdc^scid Il2rg^tm1Wjl/SzJ
018907	NOD.Cg-B2m^tm1Unc Tg(B2M)55Hpl Tg(HLA-B*0702)#Chmb/TdilJ
003355	NOD.Cg-B2m^tm1Unc Tg(B2M)55Hpl/Dvs
002570	NOD.Cg-Prkdc^scid B2m^tm1Unc/J
002452	SJL.129P2(B6)-B2m^tm1Unc/DcrJ
21 strains use this protocol