Difficulties with plasmids

Kenneth B Storey kbstorey at rideau.carleton.ca
Tue Nov 18 14:54:19 EST 1997


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
off!). 
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 



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