Problem with Plasmid Preps

Still attempting to determine what the problem is when trying to isolate plasmid DNA from a number of plasmids using the Zyppy Miniprep kit. After letting the plasmid DNA that was isolated yesterday (Problem with Plasmid Preps 5-12-14) sit in the hood for 3 hours to evaporate any excess solvent, they were ran on a gel once again to see if it made any difference.

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

Once again I don't see all my plasmid DNA. What is more frustrating is that now I don't see the sample in Well 7 like I did yesterday with the same exact sample. However, I do see from the gel that there is no loading dye present in the 7th well. This is likely a loading error on my part, or perhaps the sample add dye floated away. Letting the DNA evaporate in the hood didn't seem to help solve my problem. It should be noted that I still saw substantial amounts of EZ vision dye floating away from the well once it was loaded. It is possible still that my DNA sample may have been present there and floated away which is why it can't be visualized on the gel.


Another idea. The Zyppy Kit instructions do not include a step to dry the column before elution. I know that other kits typically include that as a step. So, this time I isolated plasmid DNA from four different overnight broth cultures in appropriate antibiotic conditions and added an additional 1 minute 13,000 x g centrifuge of the column after the last wash, but before elution.

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
Empty	Hi Lo DNA Marker	pTnMod-OKm	pEx18Tc	pEx18Tc:GW aka “pEW100”	pTnMod-Okm::Tc aka “pBE100”

By adding an additional dry step I seem to have improved my results dramatically. I clearly see plasmid DNA in Wells 4-5 and can somewhat make out DNA in Well 3. The sizes of DNA present in each well are right around where they should be based on their bp sizes. It should be noted that I still saw some sample float away from the well after loading, which maybe attributed to the faint band seen in Well 3. These are promising results and I plan to further look into this avenue (evaporation excess solvent) in the future. With plasmid DNA I can now (finally) begin my molecular work of creating a pTnMod-OTc transposon from pTnMod-Okm and also creating a knockout vector using the pEx18Tc for use in Stenotrophomonas maltophilia. I will lay out my plans for these two projects in the near future.

//EWW