10 replies to this topic

[research_freak]

Hi,

I am a new grad student. Although I have done DNA mini-preps for about 2 weeks now, I've never had problems until this morning.

I did an E.Coli transformation yesterday, incubated my plates at 37oC and found a good growth of colonies. I then scraped all the cells from the plates and performed a mini-prep using a plasmid DNA extraction kit. Since there were a lot of cells, the initial pellet was very thick. Also, when I added the lysis and neutralization buffer, there was a large amount of white precipitate.

The issue is that I got a ridiculously high concentration of DNA in both my samples, which I assume to be a contamination.

Although I worked like any other day, I wonder what are the possible sources of contamination? I took all the precautions one needs to take while performing mini-preps..

Can someone please help me?

Thanks

[HomeBrew]

How did you measure your concentration?  What do you mean by "contamination" -- DNA from contaminating organisms, or contaminants in the DNA prep that are screwing up the concentration estimate?  I would suspect the latter...

What kind of kit did you use to extract the DNA?  If you had too many cells, you would likely get incomplete lysis.  If you were too vigorous in your extraction, you would likely get chromosomal contamination.

Have you run a gel of your sample?

[scolix]

What is the conc. of the DNA samples? measure it. Also run it on gel.

[mastermi]

Yes, run it on a gel.
Remember that you can't distinguish DNA from RNA and cDNA from pDNA photometrically.

[research_freak]

I thank all of you for replying to my post!

Well, I measured the concentration using a nanodrop at O.D. 260 nm. Although the concentration should be between 100-500 ng/ul, I had a concentration of about 4500ng/ul in one tube and 2500 ng/ul in another tube!

I should have run this on the gel, but I had to hand it over to my P.I. immediately.
He was really not happy about it. Although I tried to ask other senior grad students regarding what could have gone wrong, they too said that possibly DNA must have gotten degraded and that could be the possbile reason for such a high concentration.

But I'm still shocked!

[research_freak]

I think it was contamination from the DNA prep and not from other contaminating organisms.

I used GENCATCH Plasmid DNA miniprep Kit.

[hanming86]

What's the concentration that u get actually.

Maybe since u used so much cells and maybe coz it's a high copy plasmid. then the high value might make sense.

[research_freak]

I usually get a concentration between 100-500 ng/ul.

I thought that the high DNA concentration made sense initially since I had a lot of cells. But apparently my PI. thinks otherwise.

Also, I think that since the DNA binding column has a certain binding capacity, I wonder if something went wrong at this step...

[hanming86]

I think a column can take about 20ug of plasmid not mistaken. so shouldn't be a problem.

[HomeBrew]

research_freak, on May 4 2009, 08:56 AM, said:

...they too said that possibly DNA must have gotten degraded and that could be the possbile reason for such a high concentration.

I'm not at all clear on how the degree of DNA degradation can affect the concentration...

[research_freak]

I'm not sure either, but I think that ssDNA as well as fragmented DNA have different absorption characteristics...so I wonder if that could possibly mess with the readings at 0.D. 260 nm....