Electroelution Protocol:
1. Cut about a 4inch strip of dialysis tubing (2 per sample, 1 for HMW and 1 for LMW gel
cuts) and hydrate the tubing while the gel is running in a very clean container filled with
TAE.
2. Prepare a 1% gel with large enough wells to accommodate at least 25µg of sample.
For example, if you have a sample that is 600ng/µl in 100µl of sample, you have
a total of 60µg. If you load 45µl of sample (with 5µl of 6x loading dye to run on
the gel for a total of a 50µl loading sample), you are loading 27µg (Xµg / 60µg x
100µl = 45µl, x = 27µg). You get the best yield back after electroelution when you
can load between 30-40µg.
Using 6x dye is best. Use loading dye without Orange G.
3. Skip wells between each sample on the gel, and in the first well practice by loading a
water sample with dye, to practice loading a larger volume into the gel (most likely you’ll
be loading at least 50µl depending on your concentration).
4. Run the gel for 30 minutes at 50V for the first 2 minutes, then 100V for the remainder
of the time.
5. After the gel has run, place the gel on a clean surface (clear clip board, spray with
ethanol and let dry before using). Have the dialysis tubing that is still hydrating close by
and get weighted dialysis tubing clips (white- 2 per dialysis tubing, label the clips on
tape with sample name if preparing different samples and HMW or LMW appropriately)
6. Using a clean razor blade (spray with ethanol and let dry before using). Make cuts
according to the figure below and separate the HMW cut from the LMW cut piece. The
dashed lines on the figure represent cuts, and the blue and purple are bands from the
dye, the top white box represents the well.
7. Starting with HMW cut, slice into smaller pieces as shown below and place HMW gel
pieces into HMW dialysis tubing (labeled on weighted clips, you’ll only have one clip on
the dialysis tubing at this time at the bottom to hold the gel pieces as you put them into
the open side of the tubing). It is easiest to scoop up the small pieces with a metal
spatula and assist the pieces into the dialysis tubing with a pipette tip. Once all pieces
are loaded, add ~400µl of TAE into the dialysis tubing and clip the top with the other
weighted clip. Trim the excess tubing. Make sure each tubing is labeled with the sample
name if working with different samples and whether its HMW or LMW.
(this is an example of the HMW gel piece, cut into smaller pieces, the solid lines
represent cuts)
8. Repeat step 7 for the LMW gel piece. And, repeats steps 6-8 for each additional
sample.
9. Put the tubing with gel pieces into a gel rig and run for 3 hours at ~120milliamps.
10. Label 1.5ml tubes according to each sample and whether it’s HMW or LMW.
11. After the 3 hours, remove the tubing, and transfer to clean work area. Remove top
clip and using a P-200, remove the liquid from inside the tubing and transfer to the
appropriately labeled 1.5ml tube. You should remove close to amount of TAE you put
into the tubing before running in gel rig, so ~400µl. Repeat this for each sample.
Precipitate DNA from electroeluted samples:
1. Add 1µl glycogen as a carrier to each sample. Then add 1/10th volume of 3M
NaOAc, and 2volumes of 100% Ethanol. Invert and place in the -20 freezer for 10
minutes. Spin at 13k rpm for 10 minutes in tabletop centrifuge.
2. Remove 100% Ethanol, and add 1ml 70% Ethanol. Spin at 13k rpm for 10
minutes.
3. Remove 70% Ethanol carefully and speed vac to dry the pellet.
4. Resuspend pellet in 40µl of TE. Take ODs. 260/280 value for the HMW sample
will probably be around 1.5-1.6, but the LMW sample is typically around the
desired 1.9-2.0 value. You are looking to obtain as much of the amount you
loaded in the 1% gel as possible between the HMW and LMW samples, but you
need at least 1µg of each to label.