Difficulties with plasmids
rsteinbe at etowah.ouhsc.edu
Tue Nov 18 17:30:36 EST 1997
Kenneth B Storey wrote:
> We have received several dried plasmids from collegues. Sometimes they
> are dried onto Whatman filter paper, sometimes dried onto plastic
> sheets. We attempt to retrieve the sample using TE buffers and get
> little or no DNA. We have several questions - centered around "What
> are we doing wrong".
> 1. How much plasmid should be dried onto a filter paper so that normal
> sloshing in TE buffer will remove enough to transform cells?
> We test how much DNA comes off the filter paper (spectrophoto,
> ethidium bromide,fluorometry...no matter how you try, nothing comes
> 2. Are there other ways to remove tightly dried DNA-bits?
> Do we have to electrophoresis them off? solublize with organics? face
> the blotting paper in some magical direction?
> 3. How long do dried down plasmids last on a filter paper? Any chance
> that over time either the dried bits are degraded OR over time the DNA
> just gets too tightly bound?
> 4. We have checked the competence of our cells ( and our students) by
> transforming cells with 'wet' plasmids that we make in the lab. ONLY
> the dried/resolubilized are giving problems.
> A Second ( but related) question:
> Is there a better way to 'store' critical, much-loved DNA sequences
> (such as we get from differential cDNA library screening) for the LONG
> TERM (many years) than drying down plasmids:
> Would it be possible to take an aliquot of a single stranded phage
> (containing our sequence) and then just dry *it* down in some
> protective buffer? or maybe by freeze drying the phage?
> Could the phage be re-constituted by adding buffer and stirring?
> the question may be as simple as a time line for viability:
> How long will you be able to preserve your painstakingly gathered new
> genes as:
> Storage Form: Time viable in YEARS
> 1. transformed cells stored at -70oC
> 2. dried plasmids on paper
> (assuming we can get them off correctly)
> 3.as stored phage :
> a. frozen in 7% DMSO
> b. freeze dried
> c. dried down in the cold
> d.??? storage
> thanks for any help you can give us.
> ken storey
> kbstorey at ccs.carleton.ca
We generally ask colleagues to either send DNA in a little ethanol or to
send transformed bacteria soaked onto a bit of filter paper-- both of
these have given routine success for plasmid transport. I would suggest
storing DNA as a precipitate in ethanol for long-term storage rather
than drying-- transformed bacteria slow-frozen in 15% glycerol and
stored at -70oC last for many (more than 10) years (but we generally
keep a back-up in liquid nitrogen for safety sake).
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