Cloning Tip – How Not To Expose Inserts To UV During Preparation

As mentioned in a previous post¹ there is a way that you can isolate PCR-amplified inserts from DNA agarose gels without exposure of the insert to UV.

First, create replicates of your insert by amplifying multiple PCR reactions.  I generally prepare between 3-5 replicate reactions to increase my insert yield. Prepare a 1% agarose gel + ethidium bromide. After your PCR goes to completion, cool the reactions to 4 degrees or leave it on ice. Add loading dye and load the reactions onto your gel. In the lanes indicated with the letter “L”, add your DNA ladder. I have used a 100bp ladder as an example.

Run the gel. Once completed, remove the gel from the tank and cut the gel as indicated by the red dashed line. Take the smaller portion of the gel to a dark room and visualize on an open UV box. Take a blade or scalpel and mark the gel by nicking the gel edge just above and below the band of interest (green) as indicated by the asterisks. 

Reassemble the entire gel at your bench and then using the nicks made to the gel and the ladders as a guide, carefully cut out the area where your replicate bands of interest are likely to lie as indicated by the dashed red lines.

Gel purify the gel cut-outs containing your bands of interest (green) and proceed with your cloning protocol.