The problem with open water baths and heat blocks is condensation. If the top of the tube is in the cooler ambient air, water will tend to evaporate out of the reaction and condense on the lid of the tube. This will affect the concentration of the components in the reaction and may cause problems. So best methods are ones where the entire tube is exposed to the same temperature, such as inside a pre-warmed incubator box. If you like, you could place any kind of tube holder inside the box and leave it there, so it will be pre-warmed when you place your reactions in it. Thermal cyclers with heated lids will also avoid this problem. Interestingly, for some enzymes, you can achieve greater total activity by incubation at room temperature. The rate of digestion may be lower, but the enzyme has greater stability and lasts longer. This is often useful for protocols where overnight digestion is an option.

I usually use a water bath for the digestion. Sometimes when the water bath is in use I incubate in the 37 degrees room and the quality did not differ.

I also use a water bath instead of simple incubator, But some time I use BOD incubator, since in that the uni formality of the temperature is seems to be more accurate..

Hi, Tom,

I'm using a solid-state thermostat or water bath for restriction. I've never used hybridizer like yours for such purposes, so can't directly compare efficiency. But when I need to maintain stable temperature conditions - water bath is the best.

But again - restrictases are not so picky to small temperature fluctuations.

Hi Tom,

I normally use the convection incubators at 37 degree for restriction digestion reactions and have got the expected results too. I dont see any point in using hot water bath when a normal incubator works. But yes I am curious to know why do you use a shaking incubator for such reactions?

Cheers.

I normally use 37C room for my restriction reactions. Some colleagues from Czech and Russia told me they use the pocket of their T-shirt or jeans))). I tried it and it also worked nicely.

cheers

I use water bath at 37 degrees for restriction digestion. It gives consistently satisfactory results. You may also add a few more units of restriction enzyme after 45 minutes of incubation to ensure complete digestion

Hello Tom,

I usually use the Eppendorf Thermomixer (http://www.eppendorf.com/int/index.php?action=products&contentid=1&catalognode=9832) for 60 minutes, 37°C, and at 700 rpm. Works pretty fine.

Thanks for all the opinions and suggestions, everyone. Apoorva, it isn't shaking when I use it. Konstantin, Nice low-tech, energy saving idea, although my pocket isn't big enough for a 96-well plate!

In my opinion water baths and simple incubators are similar. In water bath the heat transfer is more rapid (efficient), but after 30 min of incubation the differences are not appreciable. In my case, using Fermentas restriction enzymes, 5 min of reaction in a simple incubator is enough.

Any incubator works. You can put metal blocks that fit your tubes in there (and leave them there for that purpose), so they transfer the heat quickly to your reaction. You can also use a PCR machine and make a program for the restriction digestion.

I have used the water bath, the simple incubator, the 37° room and a thermal cycler and the results are always the same. The efficiency of the reaction depends more on the quantity of template, enzyme units and time, in my experience.

I have always used water bath at 37°C , but in my new lab they use the solid blocks at 37°C. It works either way. I started liking the solid blocks, coz it raises the temperature quickly, if you want to kill the reaction.

Hi Mr. Jones

We have used a 37°C water bath often overnight incobation and no shaking!

With the Fermentas enzymes the reaction only takes 5 min. for most digests so a water bath/PCR machine is the best if you want the reaction to be done in 5 min., a metal block with holes for tubes, in a dry incubator, would work too if it was filled with liquid, but the liquid will evaporate over time.

I use water bath at 37 degrees for restriction digestion. It works fine. But you should check the RE if it works at 37 degrees since some of them work in different degrees. But you can also use PCR device. But no need shaking.

Incubation at 37 degree in water bath is just enough. Results are almost the same with other methods. The concentration of your template and REs are important for optimal result.

incubation in water bath at 37 degree for restriction digestion....there are some enzyme which can restrict in few minutes,...but some from few minutes to few hours . it depend upon the quality of the restriction enzyme used..

A water-bath, a thermal block or a PCR machine: all should be fine for digestion w/ REs. I'm sure you've already checked the optimal conditions (template qual and quant, digest time, temp.) for the REs you're using, yet to repeat (as Mukaddes also mentioned) temp. might be a critical param: for many RE, opt. digestion temp is 37oC, yet some need, say 60oC. 2 examples (for RE from NewEngland Biolabs): recommended incubation temp for Hpy188 III is 37oC, whereas that for BsaB I is 60oC.

cheers

I use water bath set at 37oc or a dry bath set at 37oc. Any incubator set at 37oc is fine.

Yes digestion is good.

I use 37° water bath without shaking , and it works well with it. Time of incubation may vary for different enzymes but never shaking.

I routinely perform my restriction digestion on my working bench at room temperature . Well, actually it depends on the type of RE you are using. I use FastDigest from Fermentas.

If you are trying to to a fast reaction, like 5 minutes, then you may need to worry about heat transfer rates. But if your incubations are longer then it shouldn't matter very much. We usually use dry blocks for incubation of tubes and incubator for incubation of microtiter plate digestions.

Handling rectriction enzyme is too complex. Storage time in the lab, buffer quality have to taken into consideration specially when running fast reaction. However, for long incubation you only need a water bath with very limited temperature fluctuation around the required temperature for a given restriction enzyme.

the restriction digestion will work fine at 37 C at any static incubator and i think so shaking is not required.

Hi, I have tried 2 different incubations: in an oven at 37°C or/and a normal PCR cycler. I would suggest the PCR cycler as the better option.

I always use water bath and it works fine.

I always use a thermocycler just because I don't like putting reactions in water (although i know it's fine). I can control the temps much more efficiently but in the end, it really comes down to using what you have.

I typically incubate for 1-3 hours in a 37C static oven (I always double check the temp before starting a reaction). I have also done this in a water bath with the same results. Longer incubations are usually fine too (I've done overnight incubations routinely as well) so long as your enzyme does not have any star activity. You can always add BSA to help reduce star activity. It does depend on the activity and the fidelity of the restriction enzyme though. It's also important to consider what you are digesting, ie, are you digesting out of a plasmid or from a PCR product ends that might prove more difficult for a restriction enzyme to sit down on. I've had similar success with enzymes from NEB, Invitrogen and Thermo-Fisher.

I use the same incubator that we incubate bacterial plates in. I just put all the tubes in a plastic rack and place the rack in the incubator for about 1-2 h usually, and that is sufficient for the majority of restriction enzymes I've worked with. I have also used a thermocycler if I'm not going to be around to stop the reaction or a metal heat block when I'm trying to do partial digests.

The water bath has always worked well for me. The tubes are held in placed with a floating rack and left in the water bath set at the desired temperature.

Any incubator which provides required temperature will be good, dry and static ones are obviously better for long digestions. I have even used PCR machines for post-PCR digestion of my amplicons.

I use a heating block, but really heating blocks are very small water baths made for small centrifuge tubes. I like the control of temperature the heating block gives.

I usually use the PCR cycler since you can regulate the T° quickier than water bath and Oven, also, water and oven take longer to heat, PCR cycler do it in a couple of minutes and is easier to control the reaction Time

There are 3 ways...mostly all are same..

1.water bath

2.normal plate incubator

3.PCR

I used both 2 and 3 both worked good...1st also work well..

As long you are using a machine having a stable temperature the incubation of your restriction reaction will work well. In our lab we are using the static 37oC incubator we have for the incubation of E.coli plates. We are using water bath for longer (overnight) incubations. If in your lab you have the luxury to use PCR tubes 0.2ml and use the PCR machine for these kind incubations its also fine and giving the same results.

You can use water bath, incubators or PCR machines.

In principle, anything that keeps a steady temperature should work. A water bath, PCR machine or warm dry block are ideal for RE digestion because the heat transfer is more efficient with these instrument as opposed to a warm air incubator. Understand that 90% of the reaction kinetics occur within the first 5-10 minutes as soon as the reaction attains the optimal temperature.

Water bath is best

The problem with open water baths and heat blocks is condensation. If the top of the tube is in the cooler ambient air, water will tend to evaporate out of the reaction and condense on the lid of the tube. This will affect the concentration of the components in the reaction and may cause problems. So best methods are ones where the entire tube is exposed to the same temperature, such as inside a pre-warmed incubator box. If you like, you could place any kind of tube holder inside the box and leave it there, so it will be pre-warmed when you place your reactions in it. Thermal cyclers with heated lids will also avoid this problem. Interestingly, for some enzymes, you can achieve greater total activity by incubation at room temperature. The rate of digestion may be lower, but the enzyme has greater stability and lasts longer. This is often useful for protocols where overnight digestion is an option.

For restriction enzyme digestion, it is better to use PCR Thermal cycler since the temperature can be adjusted precisely rather than the water bath during digestion reaction.

I have always used an incubator maintained at 37°C for restriction digestion reactions. If you have enzymes with other requirements, like higher incubation temperatures, you could use PCR cyclers. The advantage with PCR cyclers being that you can use a wide range of temperature conditions as per the enzyme requirements. Most important factor is, having a stable temperature throughout the incubation process

In our lab we use dry bath for carrying out the restriction digestion reactions.

I have a doubt ...unfortunately i kept my thermocycler at 32 Degree overnight instead of 37 degree for restriction digestion.. do u people think the enzyme activity would work (Bam HI, Kpn I, SpeI) at 32 degree.?