- Use chemical methods when possible for DNA extractions rather than kits. Kits often cost $2-4 per sample. By chemical method in 2mL tubes, the cost immediately plummets. If you scale down your extraction into 96-well PCR plates, your reagent needs as well as your labor get minimized. Some of us just need a few PCR reactions. Depending on your organism, you may be able to do a rapid alkaline lysis method, further reducing your costs and material demands (e.g. http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CFsQFjAB&url=http%3A%2F%2Fwww.biotechniques.com%2Fmultimedia%2Farchive%2F00010%2F03344rr04_10933a.pdf&ei=98wQT7uZMam02gXiyIGECg&usg=AFQjCNF1HORNnYJQdbCmzbNYj-QGG0oJTw&sig2=lw4xwfnlpUgXNKVE3PPVrw).
- Reduce your PCR reaction volume. Most PCR kits suggest a reaction volume of at least 25uL, some as large as 100uL. I almost never do more than 10uL. If using a thermal cycler with adequate lid pressure, 6uL is possible in a 96 well plate. I now use 384-well plates and with the smaller wells (less headspace), I get good reactions of 4uL. That leaves me 2uL to run on a gel and another 2uL for capillary analysis. No waste!!
- Use reusable silicone PCR mats (e.g. http://www.phenixresearch.com/products/mpcs-3510-sealing-mat-pressure-fit-lid.asp). These things are great and I have always enjoyed less evaporation than with for instance, microseal B films. You can bleach or autoclave them if you like, but that will reduce their life. I simply throw used mats into a beaker with hot tap water for 10 minutes, rinse with RO water, and dry on a paper towel. I've never had a problem with my NTC reactions. For 384-well plates, try these: http://www.phenixresearch.com/products/smx-pcr384-sealing-mat.asp
- Use PCR plates (96 or 384) rather than strip tubes. Each plate costs about as much as 4 strip tubes last I checked. You don't have to throw out your PCR plate after running just a few reactions either. I keep partially used ones on my bench and just mark off the already used wells with a marker. When I need new wells, I peel back my silicone mat and use the unused wells, and don't bother to clean the mat until every well has been used. Still no problems with my NTCs.
- Agarose gels are reusable. Once or twice, but it can get pretty ugly. Make sure to keep spent gels intact in a sealed container (can hold numerous gels at once). Break the desired size of a gel into a flask for remelting and bring the mass back up where it should have been with RO water (if you had a 80mL gel, it should weigh ~80g). Add some new dye (e.g. ethidium or SYBR), pour, and run as always. You will notice some increased background, but for a quick gel to check some unimportant results, this works fine.
- SYBR safe can be used at 25,000X rather than 10,000X with no real loss of sensitivity.
- BigDye reactions (10uL) work fine with just 0.5uL BigDye. Add 2uL sequencing buffer, and sequence as before, or with this modified protocol I like: http://www.biotechniques.com/multimedia/archive/00003/BTN_A_000112499_O_3096a.pdf
Of course there are other things you can do, but this is all I have time for today...
Great tips! At my old lab at BYU we did the same with extractions, reaction volumes, and PCR plates. Hadn't heard of reusing gels, but that's definitely an idea. A time saving approach to gels is to make several gels at a time and then store the ones you're not using right away in a plastic bag in a fridge. You can pull them out a few days later to use.
ReplyDeleteI sometimes make multiple gels at once. I find that gels tend to acquire the shape of whatever surface they are stored on. That means if you store in a plastic bag in the fridge it will get ridges from the fridge shelf and and wrinkles you forgot to remove from the bag. I usually use a resealable glass container with a smooth bottom. Also, loss of hydration is a concern. To prevent this, keep some buffer (containing the stain you are using) in the container, preferably enough to cover all the gels you have stored. This keeps the stain "fresh" and prevents any reshaping of your gel. I have stored gels for over a month this way with no apparent reduction of detection limit.
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