Storing DNA

[Jordan Fryer]

What sort of equipment would you use to store artificial genes in vials? I thought a -20 C freezer would be good for this, but I need your opinions.

[leaking pen]

depends on how you are storing them. cut individual strands, plasmids?  

On Wed, Mar 12, 2014 at 7:06 PM, Jordan Fryer <jfre...@gmail.com> wrote:

What sort of equipment would you use to store artificial genes in vials? I thought a -20 C freezer would be good for this, but I need your opinions.

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[Jeswin]

On Wed, Mar 12, 2014 at 10:06 PM, Jordan Fryer <jfre...@gmail.com> wrote:
> What sort of equipment would you use to store artificial genes in vials? I
> thought a -20 C freezer would be good for this, but I need your opinions.
>

What exactly do you mean by "artificial genes in vials"?

Plasmids, PCR products, gDNA in buffer (TE, etc) at -20C
Lyophilized oligos can be kept at 4C

I think I have some plasmids at -80C and they've been there for many years now.

[Sebastian Cocioba]

I know invitrogen sells "genestrings" which save costs by letting u do
the subcloning. They are lyophilized fragments. Maybe that's what he
meant by artificial genes in a vial. TE buffer worked well for me. Have
some plasmids and ready to go PCR fragments at -20 for four years and
no detectable issues. I even stored some in holy water (ultra pure
depec treated) in same conditions and nothing out of the ordinary. More
important I think is dnase activity than temp. Its a very stable
molecule after all.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: Jeswin
Sent: 3/19/2014 1:30 PM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Storing DNA

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[Cathal Garvey]

I've seen differing opinions there, that when using TE you should keep
in fridge rather than freezer. The alleged reasons were:
1) It's extremely stable at 4C with TE to scavenge divalent cations anyway.
2) Freezing risks creating hypersaline (eg low freezing point) pockets
as ice crystal formation excludes TE salts, which can then fluctuate
between frozen and unfrozen states fairly easily..this can act like a
shredder in the medium term on DNA and actually decrease stability.

Point 1 is certainly true, point 2 wasn't substantiated in the source I
read, just an opinion. Given the fairly high stability in TE at 4C, I
just store in the fridge. If using ddH2O, I'd put in freezer for long
term storage.

The best "real" long-term storage is a frozen bacterial stock or
bacterial agar stab at 4C, though..provided you're *not* relying on
ampicillin or another *bacteriostatic* antibiotic to select for your
plasmid. Choose Kanamycin and you should be fine.

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[Cathal Garvey]

Aye, I've been told DNAse should be considered stable enough to survive
boiling. TE is the solution. It's not as bad as RNAse though, which is
absurdly stable; for all intents, invincible without protease treatment
or mineral acid!

On 19/03/14 19:31, Sebastian Cocioba wrote:
> I know invitrogen sells "genestrings" which save costs by letting u do
> the subcloning. They are lyophilized fragments. Maybe that's what he
> meant by artificial genes in a vial. TE buffer worked well for me. Have
> some plasmids and ready to go PCR fragments at -20 for four years and
> no detectable issues. I even stored some in holy water (ultra pure
> depec treated) in same conditions and nothing out of the ordinary. More
> important I think is dnase activity than temp. Its a very stable
> molecule after all.
>
> Sebastian S. Cocioba
> CEO & Founder
> New York Botanics, LLC
> Plant Biotech R&D From: Jeswin
> Sent: 3/19/2014 1:30 PM
> To: diy...@googlegroups.com
> Subject: Re: [DIYbio] Storing DNA
> On Wed, Mar 12, 2014 at 10:06 PM, Jordan Fryer <jfre...@gmail.com> wrote:
>> What sort of equipment would you use to store artificial genes in vials? I
>> thought a -20 C freezer would be good for this, but I need your opinions.
>>
>
> What exactly do you mean by "artificial genes in vials"?
>
> Plasmids, PCR products, gDNA in buffer (TE, etc) at -20C
> Lyophilized oligos can be kept at 4C
>
> I think I have some plasmids at -80C and they've been there for many years now.
>

--

[Koeng]

I'd say it depends on what you want to do. I always keep my plasmids/ reactions in water because I use electrocomp cells quite often (our lab uses them so often we don't actually have any TE buffer), but TE is probably better for your needs. I've just always stored my things in water and am likely to continue to do so

[SC]

I keep plasmids in 0.1 TE, which is recommended by the sequencing lab.  I've had no problems with degradation and the sequences come out OK.

[Jeswin]

On Wed, Mar 19, 2014 at 3:36 PM, Cathal Garvey
<cathal...@cathalgarvey.me> wrote:
> I've seen differing opinions there, that when using TE you should keep
> in fridge rather than freezer. The alleged reasons were:
> 1) It's extremely stable at 4C with TE to scavenge divalent cations anyway.

Hmm, DNA storage at 4C? I have not heard of that before. How long do
you think this is possible for?

> 2) Freezing risks creating hypersaline (eg low freezing point) pockets
> as ice crystal formation excludes TE salts, which can then fluctuate
> between frozen and unfrozen states fairly easily..this can act like a
> shredder in the medium term on DNA and actually decrease stability.
>

I sometimes have to use low concentrations of DNA for qpcr
(femtograms). To reduce loss to tube walls, I prepare dilutions in TE
with 100x BSA. As to long term storage, I have found that the DNA
seems to degrade and fresh dilutions are preferable. Your explanation
sounds reasonable.

On Thu, Mar 20, 2014 at 8:42 AM, SC <stac...@yahoo.com> wrote:
> I keep plasmids in 0.1 TE, which is recommended by the sequencing lab. I've
> had no problems with degradation and the sequences come out OK.
>

I will have to keep that in mind. I use 1x TE. I wonder how much of a
difference there is for long term stability. By the way, what
sequencing lab do you use?