By including crystal violet in the gel and loading buffer it is possible to visualize DNA bands as they separate during agarose gel electrophoresis. This is particularly useful for isolating DNA fragments for cloning work. The advantage of this method is that the DNA is not exposed to the damaging effects of ultraviolet (UV) light, as occurs when ethidium bromide is used for staining.APPLICATION
The improvement in cloning efficiency observed when crystal violet is used is threefold when compared with ethidium bromide and a transilluminator with max. 320 nm and tenfold in comparison with a shorter wave-length (302 nm).
- Cloning of DNA fragments (PCR products and DNA molecules digested by restriction enzymes)
+4° C to -20° CPROTOCOL
Crystal violet has a lower sensitivity than ethidium bromide. Load as much as 1 to 3 µg DNA in a slot. If less than 100 ng of the required fragment is loaded onto the gel it may be necessary to make a control with ethi-diumREFERENCES
bromide by running a second minigel at the same time.
Agarose should be prepared by adding 100 µl Crystal violet gel buffer to 100 ml gel. Add 3-5 µl Crystal Violet loading buffer to 30 µl digest or PCR aliquot and load that into the slot. For maximum sensitivity the running buffer should only just cover the gel.
After electrophoresis, place the gel on a light box to visualize separated fragments. Crystal violet is running in opposite direction as DNA, so do not run the gel for a too long time.
- 1 RAND, K. N. (1996) Crystal violet can be used to visualize DNA bands during gel electrophoresis and to -improve cloning efficiency. Elsevier Trends Journals Technical Tips Online, T40022.
500 µl loading buffer,10 ml gel buffer (100 rxns).