We have been speaking about PCR and how to manipulate this to make all kinds of DNA constructs and mutants.  However, the thermal cycler (aka PCR machine) is almost as useful!  How many things do you do in the lab that require temperature control?  Well, nearly all of them can be done in a thermal cycler.

It's Tips and Tricks Tuesday!

Back before the invention of the thermocycler, PCR was done by transferring reaction tubes from one water bath to another to change the temperature.  Imagine having to sit in front of 3 (or more) water baths transferring the PCR reaction from one, to the next, to the next, back to the first, every minute or so?  This tedium was solved by the programmable thermal cycler that can automatically change the temperature of a specified “block”.  The advent of the heated lid eliminated the need to add an evaporation blocking oil to the reaction, making the reaction easy (and much less messy) to set up and run.

But thermal cyclers aren’t just good for PCR.  We suggest using them for everything that requires controlled temperatures. 

For example:

Set your machine to dwell at 37C (or whatever temp the enzyme prefers) and you are good to go. 

If you have a double digest that includes a thermophile enzyme, like BstBI at 65C or one that likes cooler temps, ApaI at 25C, for example, program the thermocycler to dwell at the lowest temp for a period of time (1 hr is a good rule of thumb) and then automatically shift to the higher temperature (for another hour or so). 

You can even set the machine to that shift to 4C and refrigerate the reaction until you are ready for it. 

All hands off, all done automatically.

If your restriction enzyme(s) is(are) inactivated by heat, you can set the reaction to “cut and kill” by incubating the reaction at the permissive temperature and then automatically raising the temp to destroy the enzyme(s). This can be useful since some enzymes can associate with DNA during the agarose gel electrophoresis and alter the band mobility.

You can also get a similar effect by adding SDS to your gel loading buffer.

But it isn’t just restriction digests that can be done, just about any enzyme reaction to modify DNA/RNA can be done in a thermal cycler.

For example: phosphatase treatment, phosphorylation with poly nucleotide kinase, ligation, reverse transcription, (to name a few) and more, are easy to set up in a thermal cycler.

Why limit it to nucleic acids? Protein work is also easily done using a thermal cycler. 

One example:  “boil” your SDS PAGE samples in the thermal cycler, instead of using a boiling water bath.  Assuming your cycler has a heated lid, using the thermal cycler will prevent evaporation and the samples won’t “pop” open. 

You can also use the cycler to cool the sample after denaturing so that you don’t risk burns.

Trying to determine the effect of temperature on your favorite enzyme's kinetics? The thermal cycler has a perfect footprint for a 96-well or 384 well plate to do the kinetics under controlled temperatures. 

Even if you aren't concerned about different temperatures, having your samples consistently at one temperature makes separate experiments more reproducible.  

Bonus:  If your thermal cycler has a "gradient" option (where the thermal cycler creates a gradual temperature gradient across the block), then you can test multiple temperatures within the same experiment.

The 96-well footprint also makes it easy to do higher throughput westerns and homogenized assays, since it is the correct alignment for multi-channel pipettors.