Master Protocol Details



Notes:	Reaction B works better at 60 C (see 110297)
Expected Results:	Mutant =100 bp Heterozygote = 100 bp and 522 bp Wild type = 522bp

Gel Image

Image	Image file
	View Full Size
	View Full Size

Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
9485		ACC TGG AGA TTG TGC TCT GC		-
9486		GGG AAG CCA AAG AGA AAA GG		-
oIMR1084	-	GCG GTC TGG CAG TAA AAA CTA TC	-	-
oIMR1085	-	GTG AAA CAG CAT TGC TGT CAC TT	-	-

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	3.95	-	3.95
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 dNTP KAPA	0.24	0.20 mM	0.24
20 uM 9485	0.30	0.25 uM	0.30
20 uM 9486	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
20 X EvaGreen	0.60	1.00 X	0.60
DNA	2.00	-	2.00

Cycling

Step #	Temp °C	Time	Note
1	94	3 min	-
2	94	30 sec	-
3	62	30 sec	-
4	72	30 sec	-
5	-	-	Go to step 2-4, 35 times
6	72	2 min	-
7	10	-	hold

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	3.95	-	3.95
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 dNTP KAPA	0.24	0.20 mM	0.24
20 uM oIMR1084	0.30	0.25 uM	0.30
20 uM oIMR1085	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
20 X EvaGreen	0.60	1.00 X	0.60
DNA	2.00	-	2.00

This genotyping protocol is used for the following strains:

Stock Number	Strain Name
010774	B6(Cg)-Calb2^tm1(cre)Zjh/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	3 min	-
2	94	1 min	-
3	67	1 min	-
4	72	1 min	-
5	-	-	Step 2-35 cycles
6	72	2 min	-
7	10	0	Forever