Master Protocol Details



Expected Results:	Mutant = 207 bp Heterozygote = 207 bp and 330 bp Wild type = 330 bp

Gel Image

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Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
13563	-	AAG CTG GAA ATG ACC TTG GA	-	Wild type Forward
13564	-	CAC TAT CCT CCT GCC TCA GC	-	Wild type Reverse
oIMR7542	-	GGA GCT CAG GTG TCC AGT GT	-	Mutant Forward
oIMR8444	-	GCC TGA AGA ACG AGA TCA GC	-	Mutant Reverse

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
20 uM 13564	0.30	0.25 uM	0.30
2.5 ul Kapa 2G HS taq polymerase	0.05	0.01 ul	0.05
25 mM MgCl2	0.96	2.00 mM	0.96
10 mM dNTPS-kapa	0.24	0.20 mM	0.24
20 uM 13563	0.30	0.25 uM	0.30
5 mM 10x Loading Dye	1.20	0.50 mM	1.20
5 X Kapa 2G HS buffer	2.40	1.00 X	2.40
ddH2O	4.55	-	4.55
DNA	2.00	-	2.00

Cycling

Step #	Temp °C	Time	Note
1	94	2 min	-
2	94	20sec	-
3	65	15sec	-1.5 C per cycle
4	68	10sec	-
5	-	-	Go to step 2-4, 10 times
6	94	15sec	-
7	50	15sec	-
8	72	10sec	-
9	-	-	Go to step 6-8, 28 times
10	72	2 min	-
11	10	-	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 oIMR7542	0.30	0.25 uM	0.30
20 uM oIMR8444	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
006830	129-Dysf^tm1Kcam/J
013149	B6.129-Dysf^tm1Kcam/J

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	-1.5 C per cycle
4	68	10sec	-
5	-	-	Go to Step 2-4, 10 times
6	94	15sec	-
7	50	15sec	-
8	72	10sec	-
9	-	-	Go to Step 6-8, 28 times
10	72	2 min	-
11	10	-	hold