Agarose gel cutting - (Mar/18/2009 )
I am terrible at cutting my dna from an agarose gel. I dice it up pretty much and still have large pieces of agarose without dna. Do you have any advice for cutting up the gel with minimal agarose for purification? Thanks
shimshady on Mar 18 2009, 07:30 AM said:
What do you use to cut your gel?
I use a fresh scalpel blade. But I only press down into the gel. I don't drag the blade in a cutting motion to avoid damaging the glass surface of the transluminator.
I too use a fresh scalpel but I also put a piece of plexiglass in between my gel and the transilluminator to avoid breaking the glass.
I reduce the UV intensity as far as possible and switch the lamp off immediately when not needed (we have an additional lamp to have enough light in the dark room).
Practice helps a lot, I was quite bad too, therefore I cut several times DNA ladders not needed anymore.
Here some just mark with a first "preliminary" cut the area they need and then cut on the unilluminated surface besides the glass.
I do the preliminary cut as well. I have also noticed that the majority of the DNA is at the bottom of the wells, unless you mix just after loading each well. This means that you can cut the top of the slice off to minimise the amount of agarose.
The most critical thing about DNA fragment purification is UV damage and crosslinking, if you use b-agarase digestion there is not a big deal with a little extra gel. If I am gel purifying a fragment for cloning, particularly for library cloning, I load a wide prep gel well and before I expose the gel to UV, I cut 3/4 off the gel lane and never expose it to UV. I only view the other 1/4 of the lane and cut a line on the DNA band with a raze blade. Then I turn off the UV and align the two gel pieces and cut out the band under lab light so my DNA fragment used for ligation will never see the UV light. If you have problem with ligation and have trouble getting your clone, try this trick.
to make my life easier, i make use of an transilluminator without UV that still allows me to view my gel and cut them without the lights being on.
jiajia1987 on May 21 2009, 12:21 AM said:
Are u saying that u don't use UV to view Etbr stained DNA??
You can get blue light transilluminators for DNA visualisation with fluorescent dyes such as sybr safe.