1. When the cells are ready (usually 3-4 days after being split onto gelatin) wash the cells 2Xwith PBS and add 50 ul Lysis Buffer (see below) and incubate overnight at 60oC in a humid atmosphere. Use a rubbermaid container with wet paper on the bottom and a therma-seal sheet.
2. The next morning precipitate the DNA by adding 100 ul of NaCl-EtOH mix (see below) to each well. Let the plate stand on the bench 15-30 min., when the solution becomes clear you can see the precipitated DNA on the bottom of the plate.
3. Spin down the plate for 5 min. at 3500 rpm. Use a multi- or single-channel pippetteman to remove the solution. Add 150 ul of 70% EtOH to wash the wells. Again remove the EtOH with a pipetteman. Repeat the wash 2-3X. Air dry the plate.
4. Add 30-35 ul of TlowE and put at 37C in a humid atmosphere overnight. This is very important based on what enzyme you are going to use in your digest. Some enzymes will not work on the genomic DNA stuck to the bottom of your plate. You should expect between 2-10ug of genomic DNA.
5. Make digestion cocktail (shorting the water the volume of sample in TlowE) and add 50% of your sample so your total volume is 30ul. Incubate overnight at 37C. The next day load the entire sample and dye onto a gel for southern blot analysis.
Lysis Buffer: 10mM Tris, pH 7.5 NaCl-EtOH Mix:
10mM EDTA Add 750 ul of % M NaCl to 50 mls
10mM NaCl of 100% EtOH. The salt will
0.5% sarcosyl precipitate, but just mix.
1mg/ml of Proteinase K (added fresh)
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