How to store a linearized Vector? - (Nov/18/2006 )
Could a linerized vector (cut by EcoRI) be used for ligation after stored in -20C over a week?
Thank u!
as long as it is stored in TE and kept at -20 Celcius the DNA will be okay.
About 3 months ago, a labmate of mine use a vector he cut 3yrs ago, and the ligation worked fine.
About 3 months ago, a labmate of mine use a vector he cut 3yrs ago, and the ligation worked fine.
Thank you for your help!
I utilized a vector cut two weeks ago to ligate a fragment of about 700bp. A few colonies generated,and they were all religated.
I am quite sure the ligation reaction is fine. I wonder if the overhang of the linearized vector`s being degradated during the past two weeks.
And if DNA fragment eluted in TE is fine, may it not work well when it eluted in sterile water?
The EDTA in TE chelates divalent ions such as Mg2+. Mgg2+ is required for most enzymatic reactions to work.... this includes ligase, restriction enzymes and also nucleases.
So it is best to work with DNA suspended in tris-water but for storage purposes it is best to have it in TE.
As for your question, DNA stored in water may degrade with time, it's preservation is not assured.
Wheather or not the DNA degrades and how fast it will do so, would depend on your preparation skill. If you are good and clean, the distilled water will remain free of nucleases that can come from your skin, aerosol of saliva when you speak, and random bacteria that maybe on your worktop. Not so good, nuclease contamination occurs, and the clock starts ticking, the worse the contamination the faster the degredation rate.
was just wondering,if one intends to store the linearized vector for long time, maybe it's better to dephosphorylate it? 
I store it on the lab bench for a week or longer before use and its fine. As long as there is no contamination in your water that causes it to degrade it is fine. And I have had few problems with DNA stored at room temp.
I can't see the logic is dephosphorylating prior to storage....anyhow, I don't and its fine.
Well, usually cut plasmids worked in my hands even for several months (pBluescript SK+ EcoRV cut)
But if I need to get plasmids for sure and quickly, I always cut the vector fresh, not to lose the time (cutting, purification, ligation, transformation, growth, isolation of plasmids, digestion check...) until I realize that the cloning did not work.
But if I need to get plasmids for sure and quickly, I always cut the vector fresh, not to lose the time (cutting, purification, ligation, transformation, growth, isolation of plasmids, digestion check...) until I realize that the cloning did not work.
If you are storing any sort of DNA in the freezer then make sure it is kept in TE buffer. this will prevent acid hydrolysis (remember that DNA is a weak acid) occuring which will end up degrading your dna (even if your water is DNase and RNase-free!!!)