I've done mutagenesis on a couple of different plasmids and now I'm trying to get a decent amount of plasmid, using a miniprep, to send it for sequencing to see if the mutagenesis worked before I go onto doing maxi. The problem is that I get really low plasmid concentrations (ca. 10ng/ul!) irrespective of the volume of culture that I use (tried from 2, 5, 10 and even 30ml), plasmid type (pLightSwitch / pcDNA) or E.coli strain (Top10 / XL1 blue)... I noticed however that when I was repeating my experiment and checking the flow through after each step, that DNA concentration was very high after the first wash step with PB buffer... in my last plasmid extraction attempt I included 'control' samples - 5 ml of the same O/N culture eluted after using either PB/PE wash and 2x PE wash. I got from 2 to 5 times more plasmid when doing 2x PE washes instead of using PB in the first one. Has anyone had an issue like that before? I am a bit worried about the purity of my sample (the ratios were good though) but I guess the main point of PB is to improve DNA binding and PE is the wash buffer that should remove all the contaminants?
Would appreciate any advice ! (I did manage to get enough DNA for my sequencing but I still don't understand the reason behind it all and it's really bothering me! Please help!)