Problem with Plasmid Preps

Performed plasmid isolation using the afore mentioned Zyppy Plasmid Miniprep kit on overnight cultures of pTnMod-Okm in LBKan50, pEx18Tc in LBTet15, and pEx18Tc::GW in TSM Tet15 Cm20 according to the manufacturer's instruction. Laura Nessa (LN), MS, also performed the same plasmid preps along side myself in order to compare results. 

Below is a 1% agarose gel with 10 ul plasmid DNA + 2 ul EZ Vision ran at 120 Volts for 45 minutes. The description of what was loaded into each well is listed below.

Well 1	Well 2	Well 3	Well 4	Well 5	Well 6	Well 7
Hi Lo DNA Marker	EW’s pTnMod- OKm	EW’s pEX18Tc	EW’s pEx18Tc:: GW	LN’s pTnMod- OKm	LN’s pEX18Tc	LN’s pEx18Tc:: GW

I am not sure what is going on with our plasmid preps. There was little consistency between mine (EW) and Laura Nessa's (LN) plasmid preps. We both had a band of the pEx18Tc::GW show up, but only I had a band appear for the pEX18Tc. These were from the same culture sample and the same reagents. Perhaps there was excess ethanol that remained in the sample after the wash step which caused the samples to float out of the wells after loading into the well? I left the plasmid DNA sit in the fume hood for 3 hours with the eppendorf caps left open to evaporate any excess ethanol that may be present. I will rerun the same samples tomorrow and see if there was any difference.

How odd. The plasmids we are trying to extract are not larger than this kit is made for. All three plasmids are around 10 kbp in size. So far, we have used two different Zyppy plasmid extraction kits with either no plasmid appearing on the gel or it appearing with one kit, but not the other. I don't believe that the plasmid is simply just not present, as antibiotic pressure has been applied every step of the way and each plasmid conveys the resistance to the antibiotic. Maybe widespread contamination of my -80 C freezer stock?

//EWW