Greetings from the core. I have summarized some information about the preparation of PCR products for fluorescent DNA sequencing. Read on if you are interested.

Several people are interested in using PCR products for fluorescent sequencing. Cloned DNA is a very reliable template for sequencing, while PCR products are sometimes useful as templates. Depending on experimental goals, it may be helpful to clone PCR products. To sequence a PCR product, it must be pure- a single band on a gel. It is possible to purify PCR products without gel purification if nucleotides, oligonucleotides, and salts are removed. Reduction of the number of PCR cycles may give a more homogeneous PCR product. I have had good results sequencing PCR products using 20 or 25 cycles. A single substrate is essential for DNA sequencing.

A cheap and easy way to purify DNA from agarose gels was described (DEAN, ALAN D.; GREENWALD, JAMES E. (1995) Use of Filtered Pipet Tips to Elute DNA from Agarose Gels (Benchmark, BioTechniques, 18:980)). I have used this procedure along with Fisher Taq DNA polymerase and Redi-Tips (cat # 21-278-3A), both available in biology stores. I have also successfully used GenElute spin columns (Supelco) for gel purification of DNA. These columns cost about $1.50 each however, which is significantly more than a pipet tip. Using this procedure, I was able to read 500 bases of control sequence with a 1% error rate. It is more difficult to obtain sequence data after repeated (e.g. 30) Ts. This problem is less pronounced when plasmid DNA is used as template. For this procedure, use sterile solutions that are free of nucleases.

1) Perform PCR with a minimum number (20-25) of cycles.

2) Electrophorese PCR products in an agarose gel prepared with 0.5X TBE. I tested Sigma agarose (cat# A-2929). Other products may also be suitable. If a large (~100 uL) PCR was performed, it may help to precipitate DNA to reduce the volume of sample loaded on the gel.

3) Flame sterilize a flat-tipped spatula and allow it to cool. Cut the band of interest from the gel and transfer to a sterile tube for storage. This can be refrigerated indefinitely of purified (next step). The gel slice should be as small as possible.

4) Cut the tip off of a Redi-Tip and discard the narrowest end. Place the upper part into a sterile 1.5 mL tube. Add 0.05 mL of sterile water to the top of the white plug and centrifuge 5 sec. at full speed. Move the Redi-Tip to a new tube.

5) Place the gel slice into the Redi-Tip, above the white plug.

6) Centrifuge at full speed for 10 min.

7) Transfer the liquid (flow through) from the bottom of the tube to a fresh tube.

8) Estimate the volume of the flow through with a pipettor. Add 1/10 volume of 3 M sodium acetate. For example, add 0.005 mL sodium acetate to 0.05 mL flow-through. Next add two volumes (0.11 mL in this example) of 95% ethanol and vortex for 5 sec. Place the tube at -20 C for at least 10 min. Longer incubations may improve yield.

9) Centrifuge at full speed for 10 min.

10) Decant the supernatant (discard) and invert the tube on a paper towel or kimwipe to remove excess liquid.

11) Add 0.5 mL 70% ethanol to the tube and vortex 5 sec.

12) Centrifuge at full speed for 5 min.

13) Decant the supernatant (discard) and invert the tube on a paper towel or kimwipe to remove excess liquid. Completely dry the pellet in a speed vac.

14) Resuspend the pellet in a small volume (~0.015 mL) of water and determine the DNA concentration.

15) Dilute the DNA to a final concentration of 30 ng/ul. If it is already more dilute, include this information on a sequence request and give some to Scott.