How stable is double digested plasmid - (Dec/20/2015 )

Hi guys,

 

I have to repeat my cloning and hence used my previously double digested plasmid which has been stored for over a month in -20.

 

The double digested plasmid was working fine last time but this time I could not get any clones.

 

I wonder if it's because it's been cut/linearized so it's not as stable as circular plasmid.

 

Especially at the cohesive ends, do you think it'll degrade over time?

 

Thanks.

I think this depends on storage conditions, primarily whether it is stored in TE or water. Certainly the circular version will be more stable under all circumstances.

I've used double digest plasmid that's been stored in the -20C for over almost 3 years without noticeable loss of ligation efficiency.

 

I think a lot of it depends on how cleanly you prep your samples. Ensuring that you always using clean tubes, fresh water, clean agarose, clean buffers for the gel extraction, non-autodefrost freezers, etc will help tremendously. But this goes for just about everything you do in the lab.

labtastic on Mon Dec 21 15:43:12 2015 said:

I've used double digest plasmid that's been stored in the -20C for over almost 3 years without noticeable loss of ligation efficiency.

 

I think a lot of it depends on how cleanly you prep your samples. Ensuring that you always using clean tubes, fresh water, clean agarose, clean buffers for the gel extraction, non-autodefrost freezers, etc will help tremendously. But this goes for just about everything you do in the lab.

 

 

May I know if you stored the sample in TE or water? 

Meg P. Anula on Tue Dec 22 07:38:08 2015 said:

 

labtastic on Mon Dec 21 15:43:12 2015 said:

I've used double digest plasmid that's been stored in the -20C for over almost 3 years without noticeable loss of ligation efficiency.

 

I think a lot of it depends on how cleanly you prep your samples. Ensuring that you always using clean tubes, fresh water, clean agarose, clean buffers for the gel extraction, non-autodefrost freezers, etc will help tremendously. But this goes for just about everything you do in the lab.

 

 

May I know if you stored the sample in TE or water? 

 

 

I always elute my DNA in water.

 

I don't like having anything in my DNA that could interfere with downstream applications.

 

Like I said, just make sure your water and tubes are fresh, clean, DNAase-free, etc...and the DNA will be plenty stable. Good lab technique goes a long way.

This is a common point of disagreement. TE makes DNA stable, but has (minor) issues with downstream use. With 1 mM EDTA, it can chelate small amounts of magnesium from buffers. But the common 1x buffers are typically 3-10 mM Mg++, and the DNA in the reaction is typically less than 20% of the volume. You are chelating only about 0.2 mM of the Mg++ under these conditions -- hardly noticeable. The Tris makes very little difference under almost all conditions.  I'd far rather have good, stable DNA. People who use more than  20% volume of DNA in their reactions usually regret it, since contaminants such as ethanol or phenol can definitely kill reactions.

Agreed the amount of tris and EDTA is usually negligible for most downstream applications.

 

Though it's not too infrequent that I encounter applications where using >20% of volume DNA is necessary (for example, sequencing at our institution usually requires ~50% volume DNA for medium to low copy mini-prepped plasmids)

 

And since neither I nor anyone in any lab I've worked in has had stability issues when storing DNA in water at -20C, personal experience has not convinced me it is necessary. 

 

I'm used to purifying troublesome proteins, and compared to those...DNA is a rock.

 

If a student in my lab can't keep plasmid DNA stable in water in the freezer, then major improvements in their lab technique are needed before attempting to work with challenging proteins. Students gotta learn to how to skate before they can play hockey.

 

labtastic on Mon Dec 28 15:01:20 2015 said:

Agreed the amount of tris and EDTA is usually negligible for most downstream applications.

 

Agree too. I also elute my DNA in water. And stored at  -20C. But  I seldom use them for stored so long, although it could keep  ligation efficiency. I usually use fresh materials.