Protocol 22745: Standard PCR Assay - Or13c7<tm2Mom>

STOCK Or10j5^tm2Mom Or13c7^tm2Mom Or10a4^tm1Mom Or8a1b^tm7Mom Or2c1^tm2Mom/MomJ

Stock No: 027730

Version 1.3

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 = 400 bp
Heterozygote = 400 bp and 500 bp
Wild type = 500 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
mom0017		CAG AGA TCT GTG CCA GCC		Common	A
mom0018		AAG AAG GCC TTC TCC ACC TG		Wild type	A
mom0139		GAG AAG CGC GAT CAC ATG GTC CT		Mutant	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
mom0017	0.50 uM
mom0018	0.50 uM
mom0139	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
027730	STOCK Or10j5^tm2Mom Or13c7^tm2Mom Or10a4^tm1Mom Or8a1b^tm7Mom Or2c1^tm2Mom/MomJ
006638	STOCK Or13c7^tm2Mom/MomJ
2 strains use this protocol