plasmid prep question - thick white material? (Aug/08/2007 )
I am doing an E. coli plasmid prep on some transformed colonies isolated from agar media and grown up in a liquid culture. I use an alkaline lysis protocol to get the DNA out of the cells, wash with EtOH, then finally resuspend in TE. But before I do resuspend in TE, I see a clean while pellet at the bottom of my eppendorf tube that I had just centrifuged. When I do finally resuspend this pellet in TE, some of the pellet easily resuspends in TE and I can't see it anymore. The rest of the pellet doesn't dissolve so easily and looks like a thick white material, similar to what looks like a small piece of Ivory soap (not saying it is soap, but that's what it looks like!). I can break this thicker material up also and resuspend it in the TE, but I am worried this is not DNA material, but junk.
Does anyone know if this is DNA?? If it's leftover junk, how should I get it out to get the DNA only resuspended in TE?
something does not sound right here. 
It could be DNA, but cream white like soap is not how I would describe it.
could you wrirte down your protocol in detail. And point out where you see this cream white pellet?
Here is the protocol I used:
plasmid isolation from E. coli cells
1. use sterile technique - remove 1ml of transformed bacterial culture grown overnight and place in 1.5ml Eppendorf tube
2. Centrifuge at 6000rpm for 5 mins
3. Pour out supernatent and resuspend pellet in 150ul of Resuspension buffer. pipette up and down to resuspend pellet
4. Add 150ul Lysis solution. Let stand at room temp for 5 mins
5. Add 200ul Neutralization solution. Place on ice for 10mins
6. Centrifuge at 14000rpm for 10mins
7. Dispense supernatent into fresh eppendorf tubes --> at this point in the protocol, you see a lot of junk centrifuged down and the supernatent looks clear
8. Add 1000ul of 95% EtOH
9. Spin at 14000rpm for 10 mins, pour off EtOH
10. Add in 250ul of 70% EtOH, spin quickly, pour off and air dry for 10mins --> this is where I see a clear pellet and the thicker white material at the bottom of the eppendorf tube. I am not sure if the thicker material is also DNA or if not enough junk has been removed.
11. Resuspend pellet in 50ul TE buffer
I used this protocol before and it worked fine, but I don't remember if I saw the thicker pellet back then. When I ran it on a restriction digest gel back then, the plasmid DNA looked fine. I am growing up more of this plasmid this time, but see this thicker pellet every time I do this. What is it or how to get a purer cleanup of the DNA from these resuspended tubes?
err ... wemm seems to mejust the DNA !
in midipreps, i saw reguary blank things floating in my tube.
I just add more TE and these things dissolve, corresponding to high amount of DNA recovered.
Sometimes, the white stuff could be a carryover from step7.
But usually it is DNA for sure. If you want a cleaner DNA prep, use columns.
But usually it is DNA for sure. If you want a cleaner DNA prep, use columns.
I was thinking it could be a carry over from step 7 as well. The junk in step 7 will stick and run alongside the eppendorf tube. I am usually careful to get the supernatant out and not this junk. At the end of step 10, this thicker material is not sticking and running alongside the tube, but it looks similar to the junk. Could I also try using phenol chloroform maybe to get DNA only? I did the EtOH wash a few times, but still see the thicker material.
another possibility is just to do step 7 twice. centrifuge, transfer the supernatant to a clean eppendorf and centrifuge again, transfer sup again and proceed with step 8. If you still have the pellet, it is most probably DNA.
Ok, that would mean doing more EtOH washes. As a general question would repetitive washes of EtOH harm the DNA in any way?
It could also be RNA, as there is nothing here (except possibly for the mysterious buffer 1 or 2) that would contain RNAse.
This is, in general, a pretty impure way of preparing DNA, and you would expect a lot of contamination of various sorts.
Ok, that would mean doing more EtOH washes. As a general question would repetitive washes of EtOH harm the DNA in any way?
Extra EtOH washes shouldn't harm the DNA. As for the pellet, you could try adding fresh TE, after you have removed what you are sure is the DNA, and incubate for 10-15 min at 37C. Then run the supernatant on a gel.
BTW, what is the plasmid, and what cells are you using ?