Maxi prep problems - (Dec/21/2005 )

Hello all - hope you can help.

I have put standard plasmid in a new bacteria - XL10-Gold ultracompetent by Stratagene. The plasmid is just our backbone - no insert - because I need to clone into it. I got single colonies from the transformation and checked for the plasmid with a miniprep and RE digest. I took a positive colony and grew it up for a Maxi Prep (Qiagen). The Qiagen kit is new (I have only used a few of the columns) so I don't think the kit has gone bad. I got no pellet on the Maxi prep even though this backbone gives a great pellet when we are in Top10F'. I tried a second time just to be sure i didn't mess up the prep - but no pellet again.

I called Stratagene, but they were of no help. Any ideas for what is going on? Is this the bacteria or the prep?

Thanks in advance for all your help!
Kirsten

Your plasmid isn't Tet or Kan resistant, by chance? XL-10 gold has F' transposons Tn10(TetR) and Tn5(KanR), so if you were trying to select on Tet or Kan plates, you would be selecting untransformed cells. Top10F' has only Strep resistance.

Just a thought.

Actually, on further checking, XL10-Gold has Tet & Chloramphenicol resistance. XL10-Gold KanR has Tet & Kanamycin resistance, while the Top10F' cells already carry the TetR gene on the F plasmid. So, the question really should be are you trying to select with Chloramphenicol (XL10-Gold) or with Kanamycin (XL10-Gold KanR).

Check you are not overloading the column by counting the number of cells that you have. The kits are usually optimised for cells such as dh5alpha. Therefore if you have a fast growing cell type such as top10 then you should grow them for a reduced amount of time. Similarly if you use a broth different from Lb this may also lead to increased cell number. Higher than recommended cell numbers result in insufficient cell lysis and therefore drastically reduced yields. If you provide details of culture conditions, cell type, copy number of your plasmid, culture medium and your vector we can probably pinpoint what the problem is.

Hi,

you can do a quick flexi prep, before processing the Maxi prep, that is what I do when I am carrying out Mega prep at my lab. sometimes there will be no DNA at all, which is very strange this is something that I have still not understood.

Flexi prep is very simple and fast just take 1 ml of culture add the three solutions [I , II, and III] that you use for nomal pl[font=Garamond]asmid isolation, then spin at 13k for 10 min take the sup and add 0.7 volume of Isopropanol and spin at 13k for 10 min, resuspend the dna and load on the gel. you will get an idea whether the culture have DNA or not.

Hope this will help you...

I have had a similar experience where I have good results with minipreps and have trouble getting a good yield on maxipreps. I have thought that perhaps some plasmids are being lost as the bacteria divide more often . MAybe there is a toxicity issue.