Master Protocol Details



Notes:	This assay does not work well without the use of a Hotstart Taq polymerase.
Expected Results:	Mutant = 300 bp Heterozygote = 151 bp and 300 bp Wild type = 151 bp

Gel Image

Image	Image file
	View Full Size
	View Full Size

Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
11271	-	TGA TTG AGC TGG AGT TGG TG	-	Forward
11272	-	GGA GAG ATG GGT GAG CAG AG	-	Reverse

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	2.70	-	2.70
5 mM DNA Loading Dye	1.66	0.69 mM	1.66
DNA	2.00	-	2.00
2.5 mM dNTP	0.96	0.20 mM	0.96
20 uM 11271	0.60	1.00 uM	0.60
20 uM 11272	0.60	1.00 uM	0.60
5 X KAPA LR HotStart Buffer	2.40	1.00 X	2.40
25 mM MgCl2	0.96	2.00 mM	0.96
2.5 U/ul KAPA LR HotStart Taq	0.12	0.03 U/ul	0.12

Cycling

Step #	Temp °C	Time	Note
1	94	3 min	-
2	94	30 sec	-
3	58	30 sec	-
4	72	30 sec	-
5	-	-	Go to step 2-4, 35 times
6	72	2 min	-
7	10	-	hold

This genotyping protocol is used for the following strains:

Stock Number	Strain Name
013720	B6.129S6(Cg)-Ntrk1^tm2Ddg/J

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