Fast rodent genotyping

Tail clips from mice or rats of appropriate age should be snipped using sharp operating scissors and in compliance to local protocols by national and institutional regulatory organizations and placed in sterile Eppendorf tubes.

Note: This protocol has been tested using the smallest tail clips possible of ~ 1 mm. Always clean the scissors with 70% (v/v) Ethanol in between animals.

Add 20µL of Lysis Buffer and 0.6µL of Proteinase K in each sample tube.

Note: Preparing a master mix is suggested for several samples.

Incubate samples at 95Room temperature for 0h 1m 0s.

Put samples in the heat block at 98°C for 0h 1m 0s.

Spin down the samples using a mini-centrifuge for 0h 0m 30s and transfer lysate to new tubes.

Prepare PCR reactions at Room temperature as follows

Note: Preparing a master mix is suggested for several samples.

A	B
Component	Volume (ul)
Platinum master mix	10
DNA sample	1
Primers	0.4+0.4
Nuclease free water	8.2
Total	20

Note: PCR reaction may need optimization. Primers should be used at a final concentration of 0.2micromolar (µM). In this example, 0.4µL is added for each of two primers from a 10micromolar (µM) stock.

Place PCR samples in a PCR thermocycler. Set up the thermocycler with the following program using a Hot Start function.

A	B	C	D
Step	Temperature (°C)	Time	Cycles
Initial Denaturation	94	2 min	1
Denaturation	94	15 s	35
Annealing	55	15 s	35
Extension	68	20 s/Kb	35
Final Extension	68	2 min	1
Hold	4	oo	1

Note: PCR program may need optimization.

During the PCR, prepare an appropriate agarose percentage DNA gel.

Load PCR sampes along with an appropriate DNA ladder on the DNA gel.

Submit the DNA gel to electrophoresis.

Visualize gel using an appropriate device.