Strategy for Genotyping Transgenic Mice-long method

 

Day 1-Mark the ears and cut tails of mice in the evening

- Digest tails in the incubator at 55°C O/N in:

500 ml of Tail Extraction Buffer

10 ml 20% SDS

12.5 ml Proteinase K (10mgs/ml)

Day 2-Prepare genomic DNA from each tail

- Digest genomic DNA by BamHI at 37°C for O/N

- Pour a 0.8% agarose - 1X TAE - 0.5 mg/ml EtBr gel of 400 ml in the big gel apparatus with 2 combs of 24 teeth each. Cover in saran wrap

 

Day 3-Load gel with:

digested samples of tail DNA

negative and positive control tail DNA digested by BamHI OR

20 pg / 200 pg / 2 ng of plasmid control digested by BamHI

1 kb ladder DNA marker

- Run in 1X TAE electrophoresis buffer at 30 V O/N

- Label, purify and count the probe

- Photograph the gel, Turn over the gel and break DNA at 5000 mJoules

- Denature 30 min, Neutralize 30 min, Blot O/N

 

Day 4-Mark the wells on the Nytran membrane

- Crosslink the membrane

- Prehybridize 15 min minimum

- Hybridize O/N

 

Day 5-Wash the Nytran membrane.

- Place between Saran Wrap and expose in phosphorimager cassette O/N

 

 

Strategy for Genotyping Transgenic Mice-short method

 

Day 1:Mark the ears and cut tails of mice in the morning

Digest tails in the incubator at 55°C for 5 hr in 500 ml of Tail Extraction Buff + SDS + PK

Pour a 0.8% agarose - 1X TAE - 0.5 mg/ml EtBr gel of 400 ml in the big gel apparatus with 2 combs of 24 teeth each. Cover in saran wrap

Prepare genomic DNA from each tail

Digest genomic DNA by BamHI at 37°C for O/N

Day 2- Load gel early with:

digested samples of genomic DNA

negative and positive control tail DNA digested by BamHI OR

20 pg/200 pg/2 ng of plasmid control digested by BamHI)

1 kb ladder DNA marker

- Run in 1X TAE electrophoresis buffer at and 120 V for 3 hr

- Label, purify and count the probe

- Photograph the gel, Turn over the gel and break DNA at 5000 mJoules

- Denature 30 min, Neutralize 30 min, Blot O/N

Day 3-Mark the wells on the Nytran membrane

- Crosslink the membrane

- Prehybridize 15 min minimum

- Hybridize in hybridization liquid containing the denaturated probe at 42°C for 6 hrs

- Wash the Nytran membrane twice in 2X SSC, 0.1% SDS), Place between Saran Wrap.

- Expose in phosphorimager cassette O/N