Plasmid DNA mini-prep - no good isolation (May/07/2010 )

Hi everyone,
I have been using alkaline lysis method (extraction at pH 8) to extract E.coli plasmids since last year and it has always worked {Sol I has Tris-HCl and EDTA, Sol II has NaOH and SDS, and Sol III has ammonium acetate} But a couple weeks ago, after a mini-prep, my gel pictures have been showing three bands (the lowest band is the right size). When the purified plasmids were sent to sequencing, there was no generated sequences. I made fresh solution, checked my primers and everything is worked fine with clones that i have purified last year; but when I did the prep again, I got the same results (3 bands). So thinking that what's on top might be genomic DNA, I cut the plasmids out and did a gel extraction purification step before sequencing. But the yield was too low for sequencing. I am out of ideas about could be going wrong, and how to fix it . Any suggestions/comments would be very appreciated. Thanks!

I thought the plasmid should be in 3 bands: linear, nicked and supercoiled.
maybe the post here can help:
http://www.protocol-online.org/biology-for...osts/11517.html

adrian kohsf on May 7 2010, 10:04 AM said:

Thanks for the link. But I did not do any digest, so i dont think i would have linear plasmid in there. I just did a transformation, then I did the mini-prep before sending for sequencing. I have never had three bands before so thats why I don't think it should be in there.

the plasmid extraction process itself will generate the nicked and linear plasmid DNA. A picture of the gel would be nice. The nicked, linear and supercoiled bands of uncut plasmid DNA looks rather distinctive.

Elma on May 7 2010, 06:49 AM said:

Something doesn't feel right here. If I am not mistaken, the supercoiled DNA should move faster that the linearised DNA. I have a feeling that the new plasmid, may not be the right thing. Could we see a picture of your raw DNA. You might want to linearise your plasmid with a restriction enzyme to make sure your plasmid is the right thing.

But lets ignore that point for the moment. Right now you have a plasmid that is not giving you sequence data.

This could mean that there is a problem in either the sequencing reaction or the template.

Problems with the sequencing reaction, could include using too much big dye, too much template, or simply that your desired DNA sequence is not present in your plasmid, bad primer.

Template problems could include, contamination from bacteria, (some strains of E coli produce more glycoproteins than others and this can gum up the sequencing reaction), contamination from DNA extraction process itself (both PEG and phenol partially inhibit the sequencing reaction).

So where to start?

First check the template plasmid by restriction digest to make sure the desired insert is present in the plasmid. There maybe no sequence read, because there is no sequence to read.

Next, is this your first time at sequencing or is this only a single sample among many that is giving you a headache?

If this is your first time at sequencing, note that it is easy to overload the reaction. How much big dye and DNA are you using? What are you PCR conditions? The tm of your sequencing primer? Might you have labmates that could help?

If the plasmid looks good structure wise, and the seqeuncing formulations and conditions are good, you may want to use phenol chloroform to clean up your plasmid DNA. And remember to EtOH precipitate the DNA pellet after the phenol-chlorform step. And once you have the pellet, also wash it with 70% ethanol. That should remove any residue phenol.

Try this.

Make new solution of Ammonium acetate.

After one month this solution goes cuckcoo apparently due to decomposition.

My plasmid prep " resist' digestion after one month from the preparation of my solutions.

b4 that everything was ok.

changing the solution 3 made all the difference.

Thank you everyone!