shoaibarif

1. I am digesting pNL4.3 (2 ug) with NheI and Sal I. After digestion I need both fragments generated by digestion, Bigger fragment is 13kb whereas the smaller fragment is 1 kb, I am using Nucleospin Gel extraction kit to purify from the gel (as this kit can purify dna fragments greater than 10kb),
After extraction from the gel, I quantified with Nanodrop, I got only 1-2 ng/ul of both fragments, Is it normal to get such less amount of DNA after recovering from the gel ?

2. Secondly I made the ligation with 25ng vector (pNL4.3 without Sali-NheI fragment) and insert (SalI-NheI fragment) from other plasmid, and used different ratios of vector:insert like 1:2, 1;3..   Only 1;2 reaction worked but I got very few colonies gathered in the center of the plate. when I miniprep these colonies I can recover only 3-5 ng/ul of this plasmid (ligated plasmid). what is the problem here ?

3. Is the restriction enzymes digested DNA (open dna) is stable inside bacteria after transformation and bacteria can grow normally ?