Stability of cut DNA - (Jun/14/2007 )

I was running restriction digests for DNA I was cloning. I have a three-point ligation planned, and cut both insert fragments, and the vector. My stomach started hurting BAD, and it was about 2 in the morning. I felt so bad I threw the whole thing into the -20. This was about 2-3 days ago. Do you think my sticky ends are still good?

For sure. I've used them weeks later.

I usually will run my digests on a gel first though and cut the band out -- then if I have to leave, just pop the agarose piece in the fridge till the next day when I can do gel extraction.


Amanda

I have ligated cut DNA 6months old without noticable difference. A labmate ligated a vector cut DNA 2 years ago. Some lots of efficiency was noted but he got his clone. So don't worry.

I have done cloning with old digested fragments kept away for months in -20C. it works. I warm the fragments at 65C for 5 min. just to get them warmed up for ligation.

Yup, not a problem. I always keep a stock of linearized pGH19 + insert DNA in the -20 so I can easily use the Ambion mMessage mMachine kit to produce cRNA out of this. I keep this for many months before using and never had a problem. In your case I would make sure I purified the DNA product in the following weeks just to get rid of the proteins, but a RE reaction should stay stable for quite a while.

Thank you ofr the replies. How long is cut DNA stable for in 4C? How about if you cut the band out of a TBE gel and put that at 4C?

I'll just throw it into -20. No reason to keep the gel slab at 4 degrees

I will have them in -20C

Again thank you all for the replies, it is always appreciated.

I actually kept my gel slices in 4C for 3 days already. I put them in -20 until I can get to them tomorrow or the day after. I am taking the GRE tomorrow and have been stressed about it. I would in the past put DNA or gel slices at 4, because I though freeze-thawing would be worse than leaving them at 4. I reasoned that with EDTA in the TBE, they would not degrade.