Purifying Small DNA fragments with the Qiagen QG Kit
1. Prepare a 1.0– 2.0% TAE-agarose gel (see Note 1) with the comb possessing the wide or very wide teeth.
2. Load ~10 ul of 2 log Ladder in the narrow well. Then load DNA samples in the wide wells and run until the
DNA bands are sufficiently separated (~30 minutes, but best time can vary).
3. Record an image of the gel on the gel documentation system.
4. Use a razor blade on the Williams lab’s UV light box to cut out the desired DNA fragments. Try to remove,
or minimize, agarose without the desired DNA.
5. Put each agarose slab with the desired DNA fragment into a separate 2.0 ml (see Note 2) microfuge tube.
6. Figure out the weight of the agarose slab by weighing the tube + slab and subtracting the weight of an empty
tube.
7. Add 3 volumes of Qiagen QG Buffer to each tube. 1 volume equals 100 ul per .1 g of agarose, so a .4 g
agarose slab one volume QG Buffer = 400 ul and therefore you must add 1200 ul (1.25 ml) of Qiagen QG
Buffer to the snap cap tube to achieve 3 volumes.
8. Incubate samples in a 50-65oC water bath till the agarose dissolves. Mix by flicking every 5 minutes.
Dissolving the agarose usually takes 15-20 minutes.
9. Add 740 ul of the melted agarose solution to the top of a purple Qiagen column and spin the solution through
the column for 2 minutes at high speed in a microfuge. Then dump column flow through out of the collection
tube. Repeat this step until all of the dissolved agarose solution has passed through the purple column.
10. (Wash Step) Add 750 ul of Qiagen Buffer PE to the column and spin this wash solution through the column
using the microfuge at high speed.
11. Dump wash buffer out of collection tube, place purple column back in collection tube and spin one more
time.
12. Transfer purple column to a fresh 1.5 ml microfuge tube that is labeled and dated appropriately.
13. Add 35-60 ul of Qiagen Buffer EB to each column, allow sitting for 1 minute and then spinning samples in
microfuge for 2 minutes at high speed.
14. Throw away the purple columns, make sure 1.5 ml tubes are labeled properly, and close tube and store at 20oC.
Notes:
1. For DNA fragments between 0.6 kb and 4.0 kb in size, use a 1% agarose gel. For smaller DNA
fragments you should use a 1.5 or 2.0% gel.
2. If your agarose slabs are big, greater than 0.4 g, then you will want to use a 15 ml snap cap tube to
dissolve samples in.
Qualitative analysis of recovered DNA fragments on an agarose gel.
1. Set up an appropriate % TAE-agarose gel with the skinny teeth comb.
2. Run about 7 ul of the 2 log ladder in one lane. Then run 2 ul and 4 ul of your purified DNA sample in
adjacent lanes. Add 2 ul loading dye and 4 or 6 ul milli-Q to the samples to increase volume to a total of 10 ul.
3. After running the gel for sufficient time to separate DNA bands by size, take a picture of the gel on the gel
documentation system.