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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!

Liquid handling with pipettes

In Lab equipment, Pipet, Water on February 16, 2009 at 12:14 pm

Hi,

Today I would like to speak with you about liquid handling in the lab. Majority of our reactions are performed in liquids. From culturing of the cells to the specific enzymatic reactions performed, all are done in liquids. This is why we need an accurate and easy liquid handling device. We ususally perform liquid handling with pipettes.

So what is a pipette? I am sure almost everyone saw a pipette. A pipette is a device that aspirates liquids in order to transfer it from one vessel to the other. You can have a good description about general topics here.

You can have a very-very good introduction in the history of the modern molecular biology pipettes from a video by Lim Leng Hiong.

So, let’s see what “Freshbrainz” tell us about pipets:

But what kind of pipettes do we use?

The most basic pipette is a single use plastic pipette. It is not very accurate, but you can transfer liquids from one tube to a different one.

single-use-plastic-pipet1

You can use it like this:

We have a simillar pipette, a glas pipette that we use less for liquid transfer, but for removal of liquids from tubes. Usually after centrifugation steps we have a pellet and a liquid supernatant. If we want to discard the supernatant in a carefull and accurate way we use these “Pasteur” pipets. More details about Louis Pasteur here and please see a video about his work here.

So here is one of our Pasteur type, glas pipettes:

pasteur-pipet

And here is how we use a Pasteur pipette:

Of course the majority of our work is done with the so called Gilson pipettes. As our friend Lim Leng Hiong explained you these were specially designed for molecular biology work.

Below is video you can see how we handle liquids with a Gilson pipette. Please pay attention to the two stops made with my thumb. The first stop is reached when we aspirate the desired volume, while the second stop when we dispense the liquid. There is a button which is used to remove the single use tip of the pipette. So, please watch carefully the demonstration:

We have traditionally three type of pipette tips and these are differntiated by their color.

The smallest volumes can be measured with the 2 ul (2 microliter) pipette. This pipette is considered accurate between 0.5 and 2 ul-s.  The same tip is used for the 10ul pipette. We use this for volumes between 2ul-s and 10 ul-s. These pipettes are marked with gray, as shown below.

gilson-gray

The tip used with this pipetes is here:

gray-tip1

The next type of tip has yellow color traditionaly so the pipetes are marked with yellow:

yellow-heads

The same rule: P20 should be used between 10-20uls P100 between 20-100uls and P200 between 100 and 200uls.

The same tip can be used for these three Gilson pipetes, namelly these ones:

yellow-tips

The third type of this pipete is traditionally marked with blue. This is the one ml pipete. We call it P1000 and use it between 200 and 1000uls. Below is the head and the tip used for it.

blue-head

blue-tip

With this set of pipettes you can perfom majority of molecular biology reactions in the lab in an accurate way. They are not cheap, the price of one pipete is in the range of hundered dollars. They are precision instruments, so usually each researcher has his own set to use. Please pay attention to this and never use someone else’s pipete set only she or he specifically alowed it to you.

You can have a look on the usage of these pipettes on the best tutorial I have ever seen, produced by the University of Leicester here:

OK but what other alternatives so we have?

We have two very usefull type of additional pipettes. One is called the multichanell pipete, you saw it on Lim Leng Hiong’s video, and the other is the repeater pipete.

The multichanel pipet we use can have 12 or 8 chanells and you can have a look on it here:

multichanell-pipets

The range of volumes you can dispense with it can be seen here:

multichanell-range

With these pipettes the volumes can dispensed can be adjusted in steps and not in a linear way. You can see the adjustment volumes for both type of multichanell pipettes here:

multichanel steps

And here you have two videos about their usage:

The second type of very important help in the lab is the so called repeater pipete.
This pipete is able to dispense the same volume from a reservoir in a serial way.

Here is how it looks like:
repeater-pipet

The good stuff about these pipettes is that it can be used with different type of tips and it automatically recognizes the type of the tip you are using.

Here are the tips we use in general:

repeater-tips21

You can see on the next figure, that depending on the tip used the pipete is showing eighter 20 or 100 uls in the same position 1.

repeater-head

Here is a short video about how to use it:

With these pipets you can work easily in the lab. The master, the queen of lab pipettes is for sure the pipeting robot. We use a Tecan Genesis for pipeting smal volumes (5uls) in a serial way (e.g on a 384 well plate).

Have a look on this pipeting device:

robotical-pipetor-tubing

In my next post I will come up with serological pipettes and the price of the water in the lab!

Stay tuned, and lat me know if you have any questions!

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!