Purifying DNA fragments following restriction enzyme digestion or PCR using the Qiagen
PCR purification kit
1. Add 5 volumes of Qiagen Buffer PB to each reaction in an appropriately sized microfuge tube and mix.
For example a 70 ul restriction enzyme digest will get 350 ul of Qiagen Buffer PB added to it (70 ul X 5
volumes = 350 ul).
2. Label purple column for each purification and then add sample mixed with Buffer PB to the top of the purple
column.
3. Spin columns in the microfuge for 2 minutes at high speed. Dump out flow through from the collection tube.
4. (Wash Step) Add 750 ul of Qiagen Buffer PE to the top of each purple column and spin columns in the
microfuge for 2 minutes at high speed.
5. Dump flow through out of the collection tube and place column back in the tube. Then spin again for 2
minutes.
6. Label a 1.5 ml microfuge tube with appropriate name and date. Place purple column into this fresh tube.
7. (Eluting DNA) Add Qiagen Buffer EB to the top of each column. For something that will be subsequently
used in a ligation reaction or PCR I would add 35 ul of Buffer EB. If you are going to subsequently perform a
restriction enzyme digest, then I would add 58 ul of buffer EB.
8. Let columns sit with buffer for 1 minute and then spin in centrifuge for 2 minutes at high speed.
9. Throw away purple columns as the DNA of interest is now in the solution in the 1.5 ml tube. Make sure 1.5
ml tubes are labeled properly, and close tube and store at -20oC for long term storage.
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.