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Restriction Enzymes

In DNA, molecular biology, plasmid, transfection, Water on March 15, 2009 at 9:22 pm

Restriction enzymes are used to cut plasmids. We have tackled the plasmids in the previous lecture. You can have a full description about the restriction enzymes here.

As a most basic introduction I would say that restriction enzymes are enzymes of the bacteria representing a kind of immune function of the bacteria. They are present in pairs in bacteria: a DNA methylase and a restriction enzyme. They both recognize the same sequence. The bacteria is methylating its own DNA in a sequence specific manner. By this its own DNA is protected against any foreign DNA. Since horizontal gene transfer is quite common in bacteria, the bacterial cell can protect its own genetic material with the help of the restriction enzymes. The foreign DNA entering into the cell will present a different DNA methylation pattern. The unmethylated recognition sites will be cut  by the restriction enzymes and by this destroyed.

Different bacterial species have different restriction enzymes with different recognition sites (certainly each has a DNA methyltransferase, too). The nomenclature of the restriction enzyme reflects their origin. In the most trivial case the name Eco RI enzyme is informing us that it has been isolated from Escherichia coli strain R and it has been the first to have been isolated from this strain.

In the molecular biology lab we use them to cut and manipulate plasmids. They are like scissors that can be directed to specific sites in the plasmid to cleave it. With an appropriate collection of site specific cutting enzymes we can step into the very exciting field of genetic engineering.

Let us have a look to some basic usage of restriction enzymes:

You can check a good introductory video here.

In any case when working with enzymes, please use latex gloves, and keep enzymes on ice!

The unit of a restriction enzyme “U” stands for the amount of enzyme needed to cut 1microgram of plasmid with a single cutting site, in one hour, in ideal environment.

The environment of the reaction is provided by buffers. The enzymes are usually provided in a concentration of 10U/ul (10 units per microliter). The enzymes are supplied in glicerol solution and always stored at -20 C. The buffer may as well come in a 10 fold concentrated solution (10X) and it should also be kept frozen.

A typical restriction enzyme reaction is set up in the following way:

1. Check the map of the plasmid for the distribution of the cutting sites.

2. Measure the concentration of the plasmid solution by spectrophotometer. Your plasmid concentration should be in the range of 1 microgram per microliter.

3. Calculate the volume of the plasmid needed to have the required amount of product at the end. The volume of the reaction should be kept as low as possible, and should not exceed 100 ul/ reaction tube. Use sterile, DNAse free microcentrifuge (so called) “Eppendorf” tubes.

4. Plan the reaction. You should have approx 1 to 10 U of enzyme per microgram of plasmid. In the final volume of the reaction the total volume of the enzyme should be less the 1/10, because higher glicerol concentration might alter the specificity of the reaction. The buffer will be 1/10 of the final volume. Keep the final volume low (less then 100 microliters). If needed, adjust the reaction volume to the planned final volume with nuclease free water. Check the optimal temperature for the reaction. It is usually 37C, but it might differ. Check for possible star activity of the enzyme in its data sheet.

Example:

Mix the following components (ul stands for microliter):

16ul Nuclease Free Water+

1ul Plasmid solution (concentration 1ug/ul)+

2ul 10X Buffer+

1ul Restriction Enzyme (10U/ul)

Total:     20ul

5. Once the reaction is planned, start to do it: bring ice, prepare tubes, melt the buffer in your hands.

6. Pipette the required volumes of water, plasmid and buffer into the tube.

7. Add the enzyme to the tube and mix gently. Do not vortex!

8. Put the reaction into the thermostat set to the required temperature.

9. Put the enzyme and the buffer back to -20C and clean up you bench!

10. After the allocated time has  passed, stop the reaction. We are usually keeping the reaction in the thermostat for 4 hours. You can stop the reaction in several ways: by adding EDTA; by heat inactivating the enzyme at 85C for 10 minutes, or simply by freezing the tube and keeping it frozen until you purify it.

You can have a look on the applications in the video below.

Good luck!

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Water in the Lab

In Lab equipment, Water on February 1, 2009 at 5:50 pm

Hi,

Before we make the first experiment we have to discuss about some trivialities that might be different in the lab than in the outside world.

For example: water. Everyone knows what water is and I don’t want to recapitulate again the basics. You can have a real good overview here.

We use water for plenty of applications in the lab. Some of them are not specific to the lab world. Here are some examples:

3kep

Of course we use water for various lab specific purposes. The most important of these purposes is to prepare various solutions. In order to control as much as possible how our solutions will work we need a realy pure water. Tap water although is considered as pure drink water contains plenty of soluble components like: ions, colloids particles and so on. This water can not be used to prepare solutions. We use it to wash dishes but even after dish washing all dishes has to be rinsed with ion exchanged water. Ion exchaged water replaced distilled water in the last decades and stands for water that contains almost no ions at all. Distilation was used earlier to evaporate and … water and by this procedure you can get rid of the soluble salts from the water. The procedure was simmilar to the destilation of alcohool in distileries like this. The ion exchange resins are able to bind the ions from the water and produce a water that has the same qualities as distilled water has.

But how do you know if a water is pure?

It was told that you shoud use your senses: like smell it, view it, taste it. A clean water should be clear, tasteless and should not smell. But this is not enough. The easiest way to measure the presence of ions in water is by measuring its electrical conductivity. Soluble ions in the water will allow electricity to pass through the water. A really pure water is having very low conductivity.

In our lab we have a special tap for central ion exchanged water:

ioncserelt

So don’t worget, after washing lab dishes, please rinse everything at least twice with the ion exchanged water from this tap!

Can we use this water for solutions?

In some cases we could. Nevertheless due to the fact that we process sensitive biological samples like DNA and proteins we do not use this water for solutions in a molecular biology lab!

In order to prepare water for solutions we use so called “MilliQ” water. We introduce the ion exchanged water into an apparatus which is filtrating it through a replaceble cartridge. This filtrated water is free from colloids, proteins, ions and is suitable to be used in regular molecular biology solutions. Of course not for all applications! We will discuss this later. Here is the instrument that is producing the “Milli Q” water:

millipore-filter

You will find the water for solutions right in in a plastic carboy (also called demijohn) like this:

mq

You can use this water for preparing buffers for gel electrophoresis, western blot and so on.

By sterilizing it, you can make sterile solutions for cell culture applications. Nevertheless I would stronglly recommend that you should filtrate these solutions through a 0.2 micrometer filter. Majority of infecting agents (from bacterial origin) are larger than 0.2 micrometers so a sterilizes and/or filtered solution should be OK for cell culture applications.

There are some special applications that need special waters.

Two of them are RNA applications and cell culture applications for immune studies.

1. RNA applications.

While DNA can be protected quite easilly by adding EDTA as a chelating agent to the solutions (by this you get rid of the soluble Mg and other ions and you block the activity of DN-ases) RNA can not be protected like this. RN-ases are everywhere and are destroing the free RNA. That means that we have to use a special water that has no active RN-ases. Earlier we used so called DEPC treated water. Now we we use so called “Nuclease free water”. Earlier we were buying it in small 25ml bottles like this:

prom-nfw

Now we buy it in larger quantities and alliquot it. We use this water as NFW (Nuclease Free Water):

ambion-nfw

As a rule: USE ALLWAYS YOUR OWN NFW!!! Mark it with your name, and put a date when you oppened the tube.

2. The second type applications when we need an even purer water are the immunologic studies. In these cases we need a water thet is free of LPS (bacterial lipopolysaccharides, or endotoxins). The water we use for these applications is called “Embryo water” although we do not use it for embryological manipulations, it is LPS free. It is very important to alloquote it only in endotoxin free tubes, like cell freezing sterile vials.

Here is our LPS free water:

endotoxinfree

So these are the water types in our lab. We will discuss about the price of our water types later!