Recovery of DNA from Agarose Gels

3 Southern Blotting to Detect T-DNA in Tissues of Plant Material 

Digest 10 pg of plant DNA from control (noninfected leaf tissue or root tissue) and putatively transformed root culture with BamHl in a total volume 

The filter is washed gently with 100 ml 3 x SSC and air dried (DNA side up). For nitrocellulose, the membrane is baked at 80°C (under vacuum) for 45min-lh (longer times make the membrane brittle). For Hybond N, the filter can be placed on the transilluminator for 30-60 s (this should ideally be calibrated for each transilluminator at regular intervals). If Hybond N is used (Amersham), the DNA can be fixed for the membrane by treating with alkali followed by neutralization (15 min) and air drying the membrane. Other membranes can be used in conjunction with manufacturers recommendations. 

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