Genotyping  - Ear Punch DNA  (Ansel Lab 2015)

Mouse Tech 

Digest:

1. Place ear punches directly into pre-labeled PCR strips, always left-to-right.

            Don’t combine > 1 cage per strip (leave empty wells if

2. Spin briefly in tabletop centrifuge to pellet tissue (Laura’s bench)

3. Add 20 ul ear punch digestion buffer w/proteinase K per well.

            20 ul reaction = 19 ul ear digestion buffer + 1 ul proteinase K

            Master Mix for 12 strips: 1.9 ml digestion buffer + 100 ul proteinase K

4. Spin again briefly to collect contents

5. Start digest on PCR block

            PCR program Marlys/EZ type (55* x 1 hour, 95* x 5 mins, RT hold)

Lab Members

Dilute the 20 ul ear DNA digest 25x, as follows:

1.  Add 80 ul ddH20 per well via p200 multichannel, resuspend thoroughly.

2. Spin briefly to pellet any undigested bits.

3. Transfer 20 ul of diluted DNA (now 5x) to a second set of PCR tube strips.

3. Add 80 ul ddH20 per well (again) and mix again with multichannel.

This is your digested DNA.

Use 1 ul per 20 ul genotyping reaction.