Miniprep problem

[Paddy]

Hi all,

I performed a miniprep today (eager to get something I cloned off to sequencing).

Because I was so eager I thought, why not grow my XL1-Blue E.coli in YPTG media. I figured the more happy my cells are the more likely my yield of DNA will be greater. My vector of interest in this case is pET21-d (High or Low copy number, im not sure?....I should check). I did get alot of growth. So much so, that I decided to split the 10 mL overnights into two.

Anyway, I did the miniprep and speced the final product....890 ng/uL....1200 ng/uL. I know sounds dodgy right and there wasn't any bubbles. I was certainly hoping it was all my vector, and thinking that i'd grow these cells ON in YPTG again for miniprepping. However the better side of me thought to run a gel, and as suspected I got what looked like a whole lot of smearing from contaminating genomic DNA.

Another point I should mention is that during the miniprepping, the column seemed like it was clogged. I'm using promegas kit and I was extremely sure no precipitated material was put onto the column.

Any thoughts as too why im getting so much junk?
Can I use rich media to grow cells or is regular LB the only way to go?
Did i possibly have too many bacterial cells?
My cell lysis buffer (provided) had SDS crashed out of solution, so I heated it in a beaker of warm water. Could the warm buffer have anything to do with it?

Here's a pic of the gel
flickr.com/photos/37073882@N03/3413551499/

[lavendergirl]

This has happened to me before. I think you are using higher than the recommended volume of bacteria for the miniprep spin columns. Try a switch to maxiprep if you can.