Fragment size is 1-1.5 kb. Sample is already cleaned-up before restriction digestion. I just want to remove the enzyme before using the sample further for ligation.
The go to method is a phenol-chloroform wash followed by alcohol precipitation, with our without glycogen to facilitate the precipitation and keep track of the pellet. This method is quite good at removing contaminating proteins, but it can take a bit of practice to get very good yields with minimal residual phenol contamination. Residual phenol contamination may show up as a high 230nm absorbance when you quantify the DNA. For most cloning and molecular biology applications low amounts of such contamination usually isn't a serious issue. There are other methods as well, but this is the most often used non-kit method I know of.
Simply precipitate the DNA sample with two volumes of absolute ethanol for an hour or so. Centrifuge the precipitated DNA and decant Ethanol. Air dried pellet can be dissolved in desired volume of TE buffer or Water and can be simply used for ligation. You may even omit Phenol:Chloroform step. It wont matter much. For cloning the trace amount of denatured enzyme won't create problems.
A reminder: restriction enzymes user's manual usually state a denaturation temperature (and some times even a incubation time for it). For further analyses always remember to include an enzyme denaturing incubation once the cutting incubation finishes. This also prevents unspecific cutting or, in some cases, DNA degradation.
- Badges
- Science topic
- Similar topics
- Agricultural Science
- Horticulture