Why I’m not getting any band after column purification? - (Oct/12/2014 )

Recently I’m trying to construct a plasmid with desired oligonucleotide. It‘s a pET32c modified plasmid with His-Tag and some other peptide sequence with BglII and EcoRI in the MCS 3bp apart (5.6 kbp). The problem is whenever I’m digesting it with BglII and EcoRI, after column purification I’m losing my entire DNA. I’ve tried:

1. Double digestion with BglII and EcoRI- Gel electrophoresis (band okay)-column purification (No band);

2. Single cutter to check contamination with BglII, EcoRI and SphI (bands okay);

3. Single digestion with BglII, gel run, column purification (single band), then EcoRI digestion, gel run (single band), column purification (NO BAND).

4. Single digestion with BglII, then direct addition of EcoRI, then gel run (band okay)/ then column purification (No BAND).

So it looks like I’m losing the DNA in the column purification. I’ve tried the following things:

1. When gel purifying, keeping the gel in 50ᵒC for 15mins, with 4-5 times vortexing;

2. Elution with 70ᵒC heated milliQ.

3. After adding milliQ, waiting for more than 5minutes;

4. Different ration of QG:isopropanol buffer= 400:100ul/400:400ul.

5. Final centrifugation for more than 3mins at 15,000rpm.

I need suggestions. Should I avoid column purification? What are the other methods to purify digested DNA rather than column or phenol chloroform extraction?

I'd suggest that you check where your DNA is going. The Qiagen manual shows ways of collecting the column flow throughs ahd figuring out where the DNA has gone. The most likely thing is that you (or others) have forgotten to add ethanol to the PE wash buffer, which results in your DNA being washed away. This can also happen if the PE buffer is left open, so that the alcohol evaporates.