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