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Archive for the ‘plasmid’ Category

Transformation of competent cells

In DNA, GFP, molecular biology, plasmid, Resources, Restriction Enzyme, transformation on March 16, 2011 at 4:53 pm

Scientific Background

Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells into which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol. We have found that it is the best protocol. This protocol may be particularly useful if you are finding that your transformations are not working or yiedling few colonies.

In nature what happens is shown on the following two videos:

Overview

To see this in a nice lab demonstration tutorial about how transformation procedure is used, and why, watch this:

Materials

The demonstration of our iGEM protocol realized in summer 2009 is shown below:

Competent cells (we use DH5α)

DNA (this is a sample)

Ice

42°C water bath

37°C incubator

SOC (check for contamination!!)

Petri dishes with LB agar and appropriate antibiotic

Procedure

1. Start thawing the competent cells on crushed ice (we find this cells in the -70°C fridge)

2. Add 200μl competent cells and 2 or 5μl (50ng) DNA on ice

3. Incubate the cells for 30 minutes on ice

4. Heat shock at 42°C for 90 seconds water bath (not shaker)

5. Incubate for 5 minutes on ice

6. Add 200μl SOC broth (but sometimes not)

7. Shaker 2 hours at 37°C

8. (Sometimes centrifuge for 10 minutes at 10000 rpm and a few supernatant take int he dumb and suspendation the pellet)

9. Plate usually 60μl of the transformation or we make distribution 20μl and 200μl Petri dishes with agar and the appropriate antibiotic(s) with the part number, plasmid and antibiotic resistance

10. Incubate the plate at 37°C for 12-14 hours

Notes & troubleshooting

If you think another video demonstration would be needed, please go on to the next video:

More details about the procedure, with excellent links cand resource material can be found in the Molecular Biology Online Notebook.

References

1. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed.,1.25-1.28. Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, USA.

PCR or the Polymerase Chain Reaction

In animation, DNA, molecular biology, PCR, plasmid, Polymerase, Polymerase Chain Reaction, Replication, Taq on April 12, 2009 at 6:25 pm

When I first heard about the Polymerase Chain Reaction my first association was with the atomic bomb chain reaction. You know probably from your studies: the labile Uranium if receives a neutron it transformed to a stable Uranium isotope while several new neutrons are released. If these newly release neutrons meet novel labile Uranium atoms the reaction is amplified, more and more neutrons will be released until the system if is lost from control explodes in the form of an atomic bomb. If the reaction is under control we can produce heat and through this electricity in an electric plant, if the critical mass of the labile isotope is ignited with a neutron beam, it will explode.

You can have two excellent representations of the chain reaction below:

The chain reaction

But this is a blog about techniques in the molecular biology lab, so we will not deal with the fission chain reaction but we will see how a similar type of amplified reaction is produced with the DNA by specific enzymes in a reaction tube. The enzymes that can do a chain reaction are the Polymerases.

What is the function of polymerases? We have them in each of our cells. They do the most basic reaction that keeps life going on from the start of the very first organism ever. They are duplicating in a semi-conservative way the DNA in order to allow the transmission of the genetic information during cell division.

You can have an animation about DNA replication below.

What is the Polymerase doing?

The two DNA strands are connected by hydrogen bonds and code for the same information by the A:T and C:G base pairing.  The two strands are anti-parallel if we look to the double strand from one direction one of the strands will be in 5′-3′ direction and the other vice-versa in 3′-5′ direction. As an important rule we have to know that in nature all polymerases are doing the DNA synthesis in the 5′-3′ direction. The strand that can be replicated according to this rule is the leading strand. The other one is called lagging strand. The problem is that both strands have to replicated in by the same protein complex! But how can one single Replication complex produce the leading strand and the lagging strand in the same time ? We arrived to the problem of the the Okazaki fragments.

In the animation below you can find a good representation about how a single Replication complex can do the synthesis of the two different strands.

In order to speak about PCR we have to go out to trip to check some Geysers.

So let us check first the big Steamboat Geyser.

Yellowstone Steamboat Geyser

If we go closer to one of the hot springs we might see that the water is “living”, there are some algae, micro-organisms in this water. Let’s have a look:

The Yellowstone Hot Springs

These micro-organisms are living in really hot water. But if they are living, they should replicate, and if they replicate, they should have DNA polymerases!

These micro-organisms were isolated and one of them, called Thermo aquaticus (sometimes named Thermophilus aquaticus) became really famous. It has a polymerase that is used in vast majority of the in vitro DNA replication processes and in PCR.

I am sure you all have an idea already about PCR. We put all reagents needed for a DNA replication in a tube and reproduce the normal DNA replication process. So what do we need? We need a DNA template an oligonucleotide as a primer, the building blocks of the DNA (dATP, dTTP, dCTP, dGTP or in general dNTP -deoxi nucleotide tri phosphate), Mg and the Polymerase. If possible from Thermo aquaticus, which is called Taq.

There is one trick! This one trick was invented by Kary Mullis and he received Noble Prize for this single idea. The trick is, that we will not reproduce completely the natural reaction. We do not want to bother with leading strand and lagging strand and with all kind of Okazaki fragments and helicases and ligases.

The idea of Kary Mllis was that if you separate the double strand and design two oligos that will bind the two different strands but will look towards each other, than the product will be doubled. If you separate the strands by heating the solution to 95C you can repeat the reaction, and now you will have 4 copies. In the next run 8 copies and so on in each reaction you will have 2 on the power of the “cycle number” copies in a chain reaction fashion!!!

Let us have a really simple and good introduction to the whole procedure in the next two animations:

Below you can find a more fancy animation of the same procedure:

For this idea Mullis got the Noble Prize. His work changed completely the history of molecular biology. Let us check an interview with him about how he discovered PCR:

What is the practical use of this whole method?

Amplifying DNA by PCR became one of the most widely used method in a molecular biology lab. You can use it for transferring DNA from one plasmid to a different one, to introduce mutations and even to measure the amount of a specific gene in a sample. By combining it with a Reverse Transcription reaction we can measure copy numbers of RNA molecules.

Below we can see an example of how it is used in criminal justice!

In the next video you can see the workflow of DNA sequencing with a New Generation sequencing machine. What is remarkable here is that the designers of the instrument are skipping the cloning of the DNA fragments into plasmids and amplification of the plasmids by bacteria. They use micro reactors in the form of an emulsion PCR. One oligo is on a bead and the DNA binds the oligo. Each bead is fused with a small droplet that contains all the other reagents for the PCR. By this trick you will have a clonal amplification of the DNA fragments. One bead will contain on  type of DNA and you skipped all bacterial work. The result is that you can sequence the whole human genome in a couple of weeks for less the 100k USD. Or you can sequence a bacteria in a day…

Emulsion PCR in the FLX sequencer workflow

At the end of this lesson, lets have some fun and see the celebration of the PCR!

Restriction Enzyme Resources

In DNA, plasmid, Resources, Restriction Enzyme, Uncategorized on April 6, 2009 at 8:46 pm

Dear Colleagues,

Sometimes it is good to have links that cover a topic. This is why I have collected here a bunch of links that might be useful for you in your work.

You can have a good description of the methods used in restriction enzyme analysis here: Methodbook.net

If you would like to use restriction enzymes for your work, you can find a list of links of the best known restriction enzyme providers below.

New England Biolabs

Promega

Roche Applied Science: Benchmate

Invitrogen

Fermentas

If you want to start your work with these enzymes, please consult the protocol provided with the enzyme or check it at the website of the manufacturer.

Be sure you know what an isoschizomer is, what star activity is, or how you can make double digestion (details here and here).

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!

Green Fluorescent Protein or GFP

In animation, cell, DNA, GFP, molecular biology, plasmid, RNA, transfection on March 7, 2009 at 7:00 pm

Green lights in the dark

When someone first shows up in our lab, the prime goal I set up for him or her is to make “green cells” – I mean to introduce a Green Fluorescent Protein into a mammalian cell culture. In order to be able to perform this one has to know some basic molecular biology. One has to know what a cell is, what the difference is between a prokaryote and an eukaryote cell; what the central dogma is namelly that the information flows from DNA to RNA and from here to proteins is, or as it has been formulated originally and still correctly, the information flows from nucleic acids towards proteins (albeit I assume we will see exceptions for this rule, too). (You can reach a very good lecture on this topic here.)  One has to know what the difference between DNA and RNA is, in most basic approach the chemical difference is minuscule (there is a deoxyribose in the backbone of the DNA and a ribose in the RNA, there are other differences but this is the most prominent), while the results are spectacular. DNA is a quite stable molecule that can be degraded by DNAses. DNases require divalent metal ions for their activity ( usually Mg, but other divalent ions can be used too), and we can remove these ions from solutions with so called chelating agents. Most commonly we use EDTA for this task.

From practical point of view, one needs to have some backgrounds in order not to be lost in a molecular biology lab as it follows:

One has to be able to use pipettes (as seen in the previous posts), to make buffers, to know about pH, know what molarity is, and have a good basic background in maths (just enough to calculate the compositions of the buffers).

But you can perform the most basic experiment of DNA isolation even in the kitchen! At the end of this experiment you will be able to even SEE the DNA!

You can extract DNA from any cell, but the easiest way is to use some germs, like wheat or bean germs, soya germs and so on… In the following video you can see the procedure. If you do not have isopropyl alcohol (I don’t have at home for example) use regular ethanol or a strong spirit with at least 70% alcohol content!

Regarding RNA, the world of RNA is a transient world.  RNA is degraded by enzymes that can be found everywhere. RNAses can not be blocked by removing metal ions with EDTA. This makes the half life of RNA very short. Let us take the analology of the computer: DNA is like the information on the hard disk, one might have a software on the computer without using it- this is the information in the DNA. If one double clicks on its icon, the program starts, this corresponds to the transcription: information is transcribed from DNA to RNA, or the software is running, even if it is not yet in use, it is ready to get an input and process it into the output. The RNA is similarly translated by ribosome into proteins: these are the products that have been coded in the DNA. Or according to the computer analogy you create a document with the word processor software. The document is an entity by itself.  You can print it and have it. If you turn off your computer, the temporary files are destroyed, all unsaved files are deleted. So is with the RNA. RNA is carrying an information for a short period of time, it has a short half life, but can be regenerated from the DNA. These processes are explained in the following video:

Ok, so how do we make green cells? Green flourescent protein is encoded in the genome of the Jelly fish. The protein once identified can be introduced into other organisms if we isolate the DNA sequence that is encoding the GFP protein. So let’s have a look to these wonderful organisms!

Beautiful Jelly fish

The discovery of GFP protein and their mode of action changed plenty of studies in biology. The Nobel Prize for Chemistry in 2008 was given for the identification of the GFP protein and its way of action. You can see below two videos about the topic. A detailed, in depth one or below a short overview of the topic. You choose!

Giving green light to biology

Nobel Prize for GFP

After this overview I think it is time to have an experiment. We will see how you can introduce the GFP encoding DNA into a bacteria. For this we use so called plasmids as a vector. We call vector in biology a tool that is able to carry genetic information, like a plasmid, cosmid, or a virus. A plasmid is a small circular DNA that is able to self-replicate into a bacteria and to express a protein. They are responsible for lateral gene transfer in bacteria, e.g. transfering antibiotic resistance gene from one bacteria to a different one.

In the following experiment we will see the introduction of a GFP encoding DNA into a so called Agrobacterium, a bacteria that is infecting plants.

Introducing the GFP into a bacteria

Cool, isn’t it?

We can make even more complicated investigations with the help of the GFP. In the following animation it is shown the transfection process in a mammalian cell where the addressed question is if two proteins interact or not? For this they use the so called FRET or fluorescence resonance energy transfer. In order to see if the two proteins are close to each other or not, we have to use two GFP like tagged proteins with their excitation and emission wave lengths close to each other. See how it works:

Investigating protein-protein interactions with fluorescent proteins

GFP has several other applications, like tracing of migrating neurons, as seen in the following video:

Or full GFP organisms like in the following one:

If you would like to know even more about the GFP protein, please visit the best site in this topic I have ever seen, the page of Marc Zimmer, here.

I think we had even too much of GFP now, so in the next posts we will go back to plasmids…

See you!

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