DNA storage in TE for valid transgenetics purposes

[genefiend]

Hello

 

I plan on storing GFP in a -8 degree celsius fridge. how long before it degrades if The buffer used is TE? also i have heard the main problem with DNA going bad is the freeze thaw cycle from taking it out, using it, and resoring it.

 

would you say its better to store in multiple 20 ul containors, or a larger contanor that has the frozen dna shaved off with a scraping tool like a sterile razor blade.

 

genefiend.

[CharonY]

GFP? As in Green fluorescent Protein? Or do you mean DNA coding for GFP? In any case, standard procedure is to aliquot it. Generally -80° is advisable or at least -20. There is probably not much empirical data out there for something like -8°.

[genefiend]

thats what i keep reading, but i dont know if i can afford a freezer that cold.

 

what would be the difference between the coding and the actual gfp?

 

also if i heat shock it into bacteria can it then be extracted safely?

[CharonY]

GFP is the protein itself. Are you refering to transformation and what do you mean by "it"?

[genefiend]

by it i mean the gfp. you can insert it into the dna of bacteria with a heat shock therapy. you freeze it in liquid nitrogen, and then place it in warm water. theirs a 10 minute video on youtube.

 

now can the gfp be extracted from the dna without restriction enzymes?

 

i have read in some places that the some bacteria and yeast will excrete the protien into their growing media which makes it much easier to purify.

 

the reason why i ask about the bacteria is because it wouldn't require refridgeration. Then again if i had to restrict it out of their dna it may not be easier...

 

thanks for being patient with me, i know i dont know much, but i am learning quickly.

[CharonY]

Sorry, you do not make much sense right now. I assume you mean the gfp gene, but in order to put it into a cell you need to clone it into a vector first.

Since it is not clear whether it is already on a vector or not I cannot comment more on that.

So essentially you would get or create a vector with gfp include the correct promoters and transform it into a given cell (heat transformation does only work with a limited number of cell types).

The vector should be stored frozen. If you want to isolate the GFP protein, it is somewhat different, after isolation again, it has to be frozen. If anything proteins tend to die faster than DNA.