Cutting Bands and Reamplification for Sequencing

1) Scan a reverse image of the gel on a fluorimager.  (The popped open plate with the gel facing up is the reverse of how it was loaded.)  Place a print of the image under the gel plate and align using the irregularities of the gel edges.  You may find that the image is slightly (~2 lanes) wider than the actual gel.  Center the gel horizontally.

2) Cut bands with sterile scalpel blades.  Cut as small as possible to obtain only that band.  (You only need a very small piece).  Due to the slight size difference between the image and the gel, to cut bands that are on the outer parts of the gel,  cut near their inner edge of the band (toward the middle of the gel).  Lift with scalpel blade and the flat edge of a toothpick (or glass capillary tube) together.  Band will probably stick to toothpick or capillary tube. 

3) Submerge toothpick or capillary tube in microcentrifuge tube filled with 100 ul of 10mM TRIS, pH 8.0.  Make sure the band comes off in the solution.

4) Leave the tubes at room temperature overnight to elute the PCR product from the gel bands.  Then flick each tube to distribute the DNA throughout the solution.

5) In general, when reamplifying the eluted DNA, the original conditions of the PCR are “tuned down” slightly.  This is because you are already starting with a lot of template (PCR product).  Use the following guidelines.  You may find you need to adjust them slightly for each locus.  Use your original protocol with the following modifications:

            a)  Reduce the number of cycles by 5-10.

            b)  Raise the annealing temperature 2^oC.

            c)  Use a reaction volume of 50 ul to obtain enough product for sequencing.  This might not even be enough.  Sometimes I combine 3 tubes of 50 ul each to obtain enough product for sequencing.

            d)  Adjust the amount of Taq polymerase for a 50 ul reaction.  (For my regular amplifications of 25 ul, I use 1.1 units/reaction.  For amplifications of 50 ul, I use 2.2 units/reaction).

            e)  Lower the final concentration of each primer by 0.1 uM.

            f)  Lower the final concentration of MgCl₂ by 0.5mM, (but do not go below 1.0 mM).

            g)  Add 5-10 ul of template (elution) per reaction.

Last Updated: 02/27/03