Over-digestion

LA offers several advantages over digestion/solution analyses such as 1) reduction in isobaric interferences, 2)... 

Select the following tissue slides (sectioned from one tissue block) after proteinase K digestion one with suboptimal digestion one with optimum digestion and one with over digestion. 

Figure 5. Over-digested (8-minute enzymatic digestion) FFPE breast tissue stained with gene probe (red), a centromere reference PNA probe (green) and DAPI (blue). The image shows doughnut formation and uneven DNA counter stain (blue). The arrow points to a nuclei with loss of red signal. The tissue specimen has a normal 1 1 gene to reference ratio at its optimal digestion time (3-minute enzymatic digestion — data not shown).

Avoid over-digestion with the restriction enzyme. If the sequence around the site is similar to that of the cleavage site of the restriction enzyme, assume nicking due to variable specificity. Alternatively use a different restriction enzyme (or isoschizomer) or use strand separation... 

Preparation. Dissolve 100 g. of 2,4-dinitrochlorobenzene in 200 ml. of triethylene glycol, stir mechanically in a salt-ice bath to 15° (some material may crystallize), and add 28 ml. of 64% hydrazine solution by drops while controlling the temperature to 20 3° (25 min.). When the strongly exothermal reaction is over, digest the paste on the steam bath, add 100 ml. of methanol and digest further, then cool, collect, and wash with methanol. Yield 98 g. (100%), m.p. 190-192°. 

Figure 6.3 Overview over digestate treatment strategies. (Ref. 17, revised).

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