Master Protocol Details



Notes:	10X AM Buffer: 67mM Tris.HCL pH8.8, 16.6mM NH4SO4, 2.0mM MgCl2, 0.17mg/ml BSA. Dissolve in 9 ml TE, pH to 8.8, then bring final volume to 10 ml with TE (NOT water!) Filter sterilize. Add 2-mercaptoethanol to 10 mM prior to use.
Expected Results:	Mutant = ~450 bp Heterozygote = 200 bp and ~450 bp Wild type = 200 bp

Gel Image

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Gel Information:	Separated by gel electrophoresis on a 1.5% agarose gel.**

Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
oIMR1831	-	AGC AAC AAG AGC AAG CCC AAG GAC	-	Common
oIMR1832	-	GTG ACG GTG TCC GAG GAG TTG AAG	-	Wild type
oIMR5294	-	TCA TAG CCG AAT AGC CTC TCC AC	-	-

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	4.22	-	4.22
10 X AM Buffer	1.20	0.96 X	1.20
25 mM MgCl2	0.96	1.92 mM	0.96
2.5 mM dNTP	0.96	0.19 mM	0.96
20 uM oIMR1831	0.80	1.28 uM	0.80
20 uM oIMR1832	0.80	1.28 uM	0.80
DMSO	1.00	-	1.00
5 U/ul Taq DNA Polymerase	0.06	0.02 U/ul	0.06
DNA	2.50	-	2.50

Cycling

Step #	Temp °C	Time	Note
1	94	3 min	-
2	94	30 sec	-
3	63	1 min	-
4	72	1 min	repeat steps 2-4 for 35 cycles
5	72	2 min	-
6	10	-	hold**

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	4.22	-	4.22
10 X AM Buffer	1.20	0.96 X	1.20
25 mM MgCl2	0.96	1.92 mM	0.96
2.5 mM dNTP	0.96	0.19 mM	0.96
20 uM oIMR1831	0.80	1.28 uM	0.80
20 uM oIMR5294	0.80	1.28 uM	0.80
DMSO	1.00	-	1.00
5 U/ul Taq DNA Polymerase	0.06	0.02 U/ul	0.06
DNA	2.50	-	2.50

This genotyping protocol is used for the following strains:

Stock Number	Strain Name
002277	B6.129S7-Src^tm1Sor/J
002381	B6;129S7-Src^tm1Sor/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	30 sec	-
3	63	1 min	-
4	72	1 min	repeat steps 2-4 for 35 cycles
5	72	2 min	-
6	10	-	hold**