Protocol 23819: Standard PCR Assay - Asgr2<tm1Her>

129-Asgr2^tm1Her/J

Stock No: 002387

Version 1.2

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 = X bp
Heterozygote = X bp and Y bp
Wild type = Y bp

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
10631		TTA GGG CCA TCA TGG AGA AG		Common	A
10632		GTC CTG GAT CCC TTT GAT GA		Wild type Reverse	A
oIMR9535		GGG AGG ATT GGG AAG ACA AT		Mutant Reverse	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
10631	0.50 uM
10632	0.50 uM
oIMR9535	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
002387	129-Asgr2^tm1Her/J
002361	B6;129S7-Asgr2^tm1Her/J
2 strains use this protocol