Master Protocol Details

Expected Results:

Mutant = 400 bp

Heterozygote = 400 bp and 450 bp

Wild Type = 450 bp

Gel Image

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Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
mom0135	-	GCA GCT GTG GGC TTG TTC TAT GGA TCC	-	Wild type
mom0136	-	CAG TAA TAA GAC CAC AAA TGC CT	-	Common
mom0139	-	GAG AAG CGC GAT CAC ATG GTC CT	-	Mutant

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	4.55	-	4.55
5 X Kapa 2G HS buffer	2.40	1.00 X	2.40
25 mM MgCl2	0.96	2.00 mM	0.96
10 mM dNTPS-kapa	0.24	0.20 mM	0.24
20 uM mom0135	0.30	0.25 uM	0.30
20 uM mom0136	0.30	0.25 uM	0.30
5 mM 10x Loading Dye	1.20	0.50 mM	1.20
2.5 ul Kapa 2G HS taq polymerase	0.05	0.01 ul	0.05
DNA	2.00	-	2.00

Cycling

Step #	Temp °C	Time	Note
1	94	2 min	-
2	94	20sec	-
3	65	15sec	-0.5 C per cycle
4	68	10sec	-
5	-	-	repeat step 2-4 for 10 cycles
6	94	15sec	-
7	60	15sec	-
8	72	10sec	-
9	-	-	repeat step 6-8 for 28 cycles
10	72	1 min	-
11	4	-	hold

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	4.55	-	4.55
5 X Kapa 2G HS buffer	2.40	1.00 X	2.40
25 mM MgCl2	0.96	2.00 mM	0.96
10 mM dNTPS-kapa	0.24	0.20 mM	0.24
20 uM mom0136	0.30	0.25 uM	0.30
20 uM mom0139	0.30	0.25 uM	0.30
5 mM 10x Loading Dye	1.20	0.50 mM	1.20
2.5 ul Kapa 2G HS taq polymerase	0.05	0.01 ul	0.05
DNA	2.00	-	2.00

This genotyping protocol is used for the following strains:

Stock Number	Strain Name
006678	STOCK Olfr160^tm6Mom/MomJ
007766	B6.129P2(Cg)-Olfr160^tm6Mom/MomJ

Genotyping resources and troubleshooting
This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling

Step #	Temp °C	Time	Note
1	94	2 min	-
2	94	20sec	-
3	65	15sec	-0.5 C per cycle
4	68	10sec	-
5	-	-	repeat step 2-4 for 10 cycles
6	94	15sec	-
7	60	15sec	-
8	72	10sec	-
9	-	-	repeat step 6-8 for 28 cycles
10	72	1min	-
11	4	-	hold