QPCR oligo design

Designing QPCR oligos might seem complicated but there are some rules and softwares that can make it easy. In our lab, majority of data points we generate are probably measured by QPCR, so I think  it is worth to review an algorithm for desiging QPCR assays.

So, today I will describe the way I design QPCR oligos. You can have a basic intro in PCR here.
More than a year ago I switched to the UPL system, the library of probes designed by Exiqon and now marketed by Roche. The concept is quite clear, the LNA modified oligonucleotides bind much stronger to the DNA template than the average oligonucleotides. By this we can decrease the lengths of them by keeping the Tm unchanged. The UPL library consists of 165 individual oligonucleotides that are in general nine basepair long and together cover the entire genome in respect of the coverage needed to design a QPCR oligo set for any gene. You can read more about the LNA nucleotides here and here. A good website where you can calculate the Tm of the LNA oligos here: http://lna-tm.com/. The UPL system is described here.

The system allows the design of an oligo set for any DNA sequence in the UPL Design Centre. You can access the Design Centre directly here.

[caption id="attachment_581" align="aligncenter" width="468" caption="UPL Assay Design Center"][/caption]

Before we design an assay let us first look for the transcripts of a specific gene. It is very important to use annotated data, since in the annotated genomic data we have informations about possible SNP variations. This might be important, since the oligos and especially the probe should bind to an SNP free free region, because an SNP might disturb the binding of the probe to the template.

To make this data available we should use not the sequence but the transcript ID from the Ensembl.

[caption id="attachment_586" align="aligncenter" width="468" caption="Ensembl"][/caption]

At Ensembl select your species of interest, e.g. mouse and write the name of your gene of interest in the search box:



If you write into the box a gene of interest (e.g. COUP-TF2) we will see the results as it is shown here:



Here I have to click on the link of the gene and I will find the following screen: The most important info we are looking for is in the table on the top of the page:



The two transcripts of the gene are:
ENSMUST00000089565
ENSMUST00000032768.

We will use these ID-s in the UPL Assay Design Centre. First select as organism the "Mouse", and write into the box the two Ensembl transcript ID-s selected by commas. If you follow the steps the results will be like this:

Below this data you can see two links as it follows:To design an assay that would measure all transcripts select: "common assays".

The results will be given in a downloadable pdf report. Save this file and name is by the name of the gene you used as input.

The results in the pdf file look like this:



You can see that the amplicon is 95 bp long, there is no SNP in the binding regions of the primers and probes and the probe is closer to one of the primers. There is an SNP in the gene that was avoided by the program. You can have an SNP even in the amplicon, unless is not in the binding site of the primers or the probe.

Before I order the oligos, I usually test them with e-PCR on the UCSC Genome Browser.

[caption id="attachment_608" align="aligncenter" width="468" caption="UCSC Genome Browser"][/caption]

Select the PCR view and paste the oligos into the given locations. Select the genome, the assembly and the target as "UCSC Genes"(If you used a genomic sequence for design use the target: "genome assembly").

If you have a hit, click on the link provided and the results will be represented in the genome as seen here:



The oligos are intron spanning and in the right location. Order the oligos in an HPLC pufied form in the lowest available scale for the first try. Be aware that according to the experience of several groups, and my own experience too,  only 2/3 of the UPL assays work without further optimization. This means, if you want to be sure from the first you better try two, three different assays for the same gene. The UPL Design Centre will generate several primer-probe sets and you can retrive these results too. Since the UPL library is given and one or two of the three ordered assays will work, it this worth trying three from the beginning!

In general the rules for a good QPCR assay:

1. The amplicon should be as short as possible (60-70 bp is ideal, but should be shorter than 100bp).

2. The Tm of the oligos should be around 60C, while the Tm of the probe 10C higher.

3. The distance between the the oligo and the probe should be as small as possible for a better exonuclease activity of the Taq polymerase.

4. The GC content of the two oligos should be as close as possible.

5. The number of GC bases in the last five nucleotides on the 3' ends of the two primers should be identical (if possible).

6. Select for oligo sets with week internal bonds (less than four H bonds in the same conformation).

7. Avoid primer dimers that could produce artefacts due to the 3' elongation of one of the primers.  The same for internal conformations.  See below:

8. Verify the oligos with e-PCR on the UCSC Genome Browser. The test should give one single hit!

9. If possible use annotated sequences to avoid the SNP effect.

10. If you are looking for genes (cDNA measurement) use exons that are common for all transcripts variants (or use the "batch assay-common assays" in the UPL Assay Design Centre)

Good luck!

What you always wanted to know about viruses

Considering that swine flu is here everywhere and many became interested in viruses, I compiled here a few videos that might give you an update about viruses, virology, vaccine production and how a vaccine acts. Feedback and questions are welcome at

balintblaszlo(at)labtutorials(dot)org

Regards,

Balint.

Where are the swine flu cases?

See actual status here

or check it in GoogleMaps here:

[googlemaps http://maps.google.com/maps/ms?ie=UTF8&hl=en&t=p&msa=0&msid=106484775090296685271.0004681a37b713f6b5950&ll=32.639375,-110.390625&spn=15.738151,25.488281&output=embed&w=425&h=350]
An overview of the status by 20090430:



How does influenza spreads and what happens with it in our body?


The life of a virus


The structure of viruses


Vaccine production(description and images).

Vaccine production at Sanofi (video).

How does the vaccine act?


Injection of the vaccine


More about pandemics, and vaccination here.

How microarrays can be used for rapid characterization of viruses: here.

Stay tuned!

Bird flu solved, swine flu arrived!

The bird flu solved by vaccines, now comes the swine flu.

Pandemics shaped the human population in past and probably in future. One of the most destructive influenza pandemics in the last century was the Spanish Flu which caused the death of 25-40 million people. More info about the pandemics here. The very last thread to cause a pandemic is the virus called A/H1N1, or swine flu, or Mexican flu.

Rapid diagnostic tests will be probably be PCR based. A very interesting microarray based virus characterization tool is described here.

Influenza vaccines are produced by quite a lot of manufacturers in the world. You can have a list of the manufacturers here.

A small Hungarian company is a word leader in vaccine development. Omninvest was one of the first who produced the anti H5N1 vaccine. It is good to draw the conclusions of the Bird Flu vaccine development, a detailed overview can be found here.

Today Omninvest announced that they made all the preparatory steps needed to start to develop a Swine Flu vaccine. Once the sample virus arrives they can start the development of inactivated viruses that can be used for the development of the Swine flu vaccine. While US based CDC researcher Ruben Donis announced that the "reference strain" would be available at the beginning of May, the Hungarian news agency MTI released the information that samples that could be used for further development migh arrive in days.

For more details about the life of a virus and vaccination check this.

Dear All,

In this changing world we all feel the pressure to find novel ways to increase our efficiency and impact. Recently I just came in contact with Howard Wolinsky a freelancer scientific and medical journalist. The way how Howard changed the pace of his work seems to reflect the "flat world" paradigm. We will discuss about journalism, being a freelancer, scientific journalism, and the impact of web based networking via Facebook, LinkedIn, Twitter and so on.

So let us have a short interview with Howard Wolinsky:

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LTB:  After working so many years as an employee, you decided to become a freelancer. What was your strongest motivation to make this step?

HW: I started out as a newspaper reporter in a small town, Kankakee, Ill., USA, 1970, covering mental health (there were two large mental institutions in town) and local government.

But I had my eyes on bigger things. I wanted to be a health/medical reporter for a major daily.

My colleagues often described me as "entrepreneurial." That meant, I freelanced and they didn't for the most part.

I started to freelance articles to the Associated Press and United Press International. I did a story for the Chicago Daily News--an historic newspaper where Carl Sandburg, the poet, and Ben Hecht, the playwright, who authored "Front Page," had worked--on a mental health topic. I was offered another assignment.

But the Kankakee Journal nearly fired me for writing for a "competitor."

But I kept freelancing throughout the years. Some years, as I moved on to other papers, including St. Petersburg (Fla.) Times, Florida Today (then in Cocoa, Fla.) and Chicago Sun-Times—my freelance wages matched or exceeded my base pay.

I considered leaving to be a full-time freelancer a couple times. But one of the problems in the US is health insurance. One time, on the verge of freelancing full-time, I contracted pneumonia and was out for a couple weeks. My pay at the Sun-Times continued. But had I only had freelance, I would have had financial worries. No work, no pay as a freelance.

In the intervening years, my wife returned to work and could offer us insurance coverage.

As the economics of the newspaper business deteriorated in general and at the Chicago Sun-Times in particular, I had an opportunity to take a buy-out after nearly 27 years on the job as a health and tech reporter. As I described in an article in GaperBlock.com, I had a flash of insight on how I could take a year's pay as "seed money" to start my own freelance business.

I left on a Friday in January 2008. The next Monday I had my first assignment.

But in fact, I unconsciously been preparing for this move since 1970.

A friend once advised me that I shouldn't just have one job but multiple ones. That's what I did last year. In addition to freelance writing, I diversified. I went in new directions. I networked.

I became the US blogger for Skype. I taught at Medill Graduate School of Journalism at Northwestern University. I wrote for university/medical school magazines, such as Cleveland Clinic, Northwestern Memorial Hospital, Roswell Park Cancer Center, Howard Hughes Medical Institution, University of Illinois at Chicago. I wrote for BusinessWeek, more general, non-science stories. (Check out my story in BW on David Axelrod, President Obama's advisor.) I was invited to contribute to Huffington Post, which doesn't pay (yet) but gave me a chance to stretch and write in new areas. I wrote for my former competitor, Chicago Tribune. I wrote some articles for Venture Capital Journal.   I did some consulting. I started doing more for my friends at EMBO Reports. I did a number of stories for WebVet.com. On and on.

And as busy as I was, I spent two months traveling. My wife and I celebrated her birthday and the Inca New Year at a yoga retreat in Peru (we were remarried by a shaman at Machu Picchu). The BBC paid my way to UK to appear on the Coast program. We went to Paris on the train for a day. Took a couple trips to Northern California and one to Florida.

LTB: You are doing plenty of different type of activities: you write books, you are a blogger for Skype, US correspondent for EMBO Reports, you are teaching and this is only the tip of the iceberg. How do you manage to keep your focus? What is your secret of productivity?

HW: Some days, I do feel that I have taken on too much. I am a fast writer, which helps. I also have turned away work if I didn't find it interesting, but I always tried to steer the work to a friend. It's important to network. (One of the things I did everyday after I left my full-time job was spend an hour a day building my network at LinkedIn, Facebook and now Twitter. Jobs have come my way because people have found me in LinkedIn and Facebook--even though I can be readily found through a Google search.)

LTB: You are located in Chicago but a substantial part of your activity is targeting a much wider audience. Do you see a trend in this? Is the world really becoming flat? From practical point of view do you think people need a strong local connection network and mix this with a web based business or you see that targeting people trough the web could be enough in itself?

HW: I always told my kids to think globally because that's where the work will be. I also told them to become fluent in another language. (I am working at that now. I am taking Spanish via Skype with a tutor in Guatemala.)

The Web offers the tools to reach out and be a world citizen/worker. I ran across my phone bill from a few years ago, I cut US $2k off by using Skype. Before I became a blogger for Skype, I wrote an article for the ScienceWriters network urging my colleagues to use VOIP to expand their network of sources and also clients. Everyday now, some 350,000 people sign up for Skype. Over 400 million already have. It's amazing--and I'm not just saying that because I am a blogger for Skype.

Seemingly silly social media, such as Twitter, are and will become increasingly important. Some people I run into seem to feel they already take up too much time doing e-mail. They reject doing Facebook and Twitter. I even talk to young people who feel that way. Some see it as an invasion of privacy. They feel teched out. Get over it.  I say give it a try. Facebook and Twitter can be about more than what you had for breakfast.

LTB: As a freelancer, can you plan your work for longer time periods (I mean more than 3 to 6 months)?

HW: I do the best I can to structure my time so I can count on a revenue stream. So far, I can rely on Skype and Medill as a base. Everything else is gravy. It's hard to know what's coming. I am helping develop a new magazine now, but this won't last. I have a line on a potentially lucrative consulting job in the fall, but I can't count on it. Clients come and go.

LTB:  What was the most important for you to have a successful transition from employee to freelancer?

HW: In the US, make sure you have health insurance. I had the advantage of working in the field for decades. It would be different if I were totally starting out. Networking is vital. Some writers I know are great networking to cover a beat, but may don't have a clue on networking in the writing business world. They're going to have to learn.

When I decided I was leaving my job, I wrote down every e-mail address I had and sent out notes announcing my departure. The networking helped. Contacts came up with leads that turned into jobs.

I had belong to the National Association of Science Writers for 25 years. Never went to a meeting. Until last year. I walked away from my first meeting with a lead on the teaching position at Medill. I have joined several other groups and have attended meetings. You never know when one thing leads to another.

Help others find work. They may return the favor.

Use LinkedIn, Facebook and Twitter.

LTB: With your experience now, at what stage of a career would you recommend younger people to start their preparation to become a freelancer?

HW: Maybe we'll all be freelancers in the future. The 27-year job like I held seems to be disappearing with the daily dead tree newspaper.

On my first job in 1970, we used manual typewriters. Soon, we switched to IBM electrics. We typed a code on the stories and old-fashioned pressmen--the guys in the paper hats--fed the stories into scanners. We became the first computer generation.

But I'll never forget one of the columnists, Gil, couldn't adjust to the electric typewriter. He became frustrated. He gave up. He was like the film stars of the silent era who couldn't make the transition to the talkies. Gil retired in his '50s.

Gil is an object lesson for us all. Unless you just want to go fishing full-time--maybe for good for you, but not me: Be open to change. Embrace new technology. Be excited about what you're doing. Keep learning.

LTB:  But where is journalism going these days? I see that classical media is shaking and journals are changing habits. So journalism is changing too. But my specific question here is, how will scientific publishing change? We see the waves of Open Access Journals Movement (PLOS and the others) but these are still good classical peer reviewed journals. Do you see any novel type of media that could replace journals? What is your opinion about JOVE (Jounal of Visualized Experiments). Could this be a new type of publishing?  Could someone build a scientific blog and stop counting the Impact Factors but start to count visits and links? Could this kind of change happen?
Too many questions again.
Maybe let's summarize it in one single question:
Where is scientific journalism going?

HW: Good question. But I am no visionary on scientific publications, let alone lay publications.

Maybe 10 years ago, I saw a collision coming. I saw how all lay media were becoming one. I heard audio clips on the Wall Street Journal site, and saw text stories on TV websites and now video clips on print news sites.

I even have become a bit of a video reporter. I always try to do video interviews via webcam for my stories on the Skype blog. People have different ways of accessing/inputting information. The more choices you can give them, the better your odds of reaching them.

I also took a visual story telling class to learn what I could about doing video for the Web.

That said, I am not familiar with the video project you mentioned. I'll have to take a look.

All media seem to be going through a revolution, seeking new models and new ways to find and pay their way.

The students in my science/health writing class at Medill/Northwestern—the next generation of media— are required to do text stories. But also to do video and interactive graphics. They need to be flexible and masters or at least knowledgeable about all media.

I hope text--at least web-based articles, if not dead trees--will survive. Video story telling can be compelling. But I'd be skeptical that really complex stories can be told that way. Maybe I sound like a sentimental traditionalist, but I hope the word survives in print or on the web, for lay as well as scientific publications.

Thank you very much for your time and effort to share your ideas with us!

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You can have a deeper insight in Howard's work on the following sites: here,  here, here and here

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

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).

Hunting Viruses

If it comes to speak about the future possibilities of molecular biology, it is worth keeping an eye on the medical applications. And if you think about the most peculiar infection agents you should for sure think of viruses also. What is a virus? Of course we all know what a virus might cause to us, like a simple respiratory infection. These are usually caused by viral infections that later are super infected with bacteria. I had a professor who tried to explain us what a virus is. It skips almost any definitions. We can not be sure if we could consider a living thing at all!!! At the end he told us in a laconic way: a virus is a VIRUS! Nothing more.

If we try to find them it is good to know, that they were discovered through the observation that you can transfer an infection from one cell culture to the other even after filtrating the solution through a filter with 0.4 micrometer holes. That means that no bacteria can bypass this filter, but infections can be transferred with this solution. The firs experiments were done in order to monitor these infections, to see that after infection there was a "clean window period", a period when the infections agent disappeared from the cell culture. After this window the virus reappeared and the supernatant solution had infections properties again.

Today we know plenty of details about viruses. There are basically two flavours of them DNA and RNA viruses. So that is an important point! because we have plenty of molecular biology tools that allow us to characterize nucleic acids. One of the most complex tool from this series is the DNA microarray. As one of my students pointed out last week, in the next video from TED, we can have a wonderful presentation about how these tools can be used in a fast and relatively easy way to get a deeper insight in the world of viruses. As a perspective the video shows us some excellent diagnostic applications that will be probably used to develop state of the art diagnostic tools in the next couple of years.

So, let us see how it works!



More info about the viruses and vaccination, here.

Restriction Enzymes

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

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!

Using Serological Pipettes

Dear Colleagues,

I promised you to give an update about serological pipettes.

We use serological pipettes when we want to manipulate (to move) liquids that are in the range of 5 to 25 ml. Smaller volumes than 5ml can be measured with Gilson type pipettes, while for larger volumes than 25ml we use measuring cylinders.

Serological pipettes have a dispensable graduated tube, and a filter that is not allowing contamination with any particles from the air. The pipettes look like this for example:

[caption id="attachment_199" align="aligncenter" width="468" caption="serological-pipette"][/caption]

They can be charged, have a button to move liquids up and one for release the aspirated liquids.

We use sterile, single packed pipettes in three different ranges: up to 5ml, to 10ml and to 25ml. They have different color codes:

[caption id="attachment_201" align="aligncenter" width="468" caption="sterile-serological-pipettes"][/caption]

The same from their back:



Please have a look to my demo of how to use them:



Settings of a typical serological pipette can be seen below. You can adjust the power of the pump that enables you to move volumes as small as 1/10 of ml. And you can switch between "drop wise" and "blow out" mode. You use "drop wise" mode when you do not want to disturb the cells on the bottom of a culturing dish.

[caption id="attachment_205" align="aligncenter" width="468" caption="head-of-serological-pipette"][/caption]

A detailed demo about serological and other similar type of pipettes:



What about using Gilson type pipettes in the cell culture lab? We have sterile, pyrogenic free tips for the cell culture lab. This box contains for example certified DNAse, RNase and Pyrogen free tips. Each tip has an individual filter insert! You can use them for probably any protocoll in a standard molecular biology lab. Don't forget, they are not cheap at all...

[caption id="attachment_207" align="aligncenter" width="468" caption="barrier-tip-box-1ml"][/caption]

[caption id="attachment_209" align="aligncenter" width="468" caption="barrier-tip-1ml"][/caption]

Smaller volumes can be measured with smaller barrier tips:

[caption id="attachment_221" align="aligncenter" width="468" caption="barrier-tip-10ul"][/caption]

I think we should go to the cell culture lab in one of our next post!

That's all for today, and let me have your feedback!

Plenty of good basic info regarding laboratory work is described <a href="">here.


Cheers,

Balint.

Why Molecular Biology?

At the very end you might ask why is the life in a molecular biology lab so interesting?

We discussed about water, pipettes and we will go on with several topics, but at the very end there is a wonderful, miraculous world. Each cell in our body and each cell in any living organism works based on the same principles. Information is stored, processed and replicated in cells.

If we could have an insight into these processes we could better understand what is life. Yes, I think this is still a question! What is life? How can you explain the abundance seen on every cubic centimetre of the surface of this planet?

Instead of giving a flat answer, let us look to the best animation I have ever seen about THE INNER LIFE OF THE CELL!

Here it is:

Liquid handling with pipettes

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.



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:



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.



The tip used with this pipetes is here:



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



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:



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.





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:



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



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:



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:


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:



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.



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:



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

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:

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:

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:

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

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:

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

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:

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

What?

Dear Colleagues!

I have decided to make an online collection of the basic (and not so basic) techiques we use in our lab. This is a (hopefully) classical molecular biology lab located in Europe, Hungary, more close in Debrecen. I will present you the lab and environment later.

The idea is to describe these techiques, make a pdf version of the protocols we are using and in some of the cases to upload videos about these techinques.

Any feed-back is welcome at:



Please stay tuned,

Balint

Dear all,

The setup seems to be quite clear.

Every semester we have at least five students coming into our lab to get involved in molecular biology techniques. Untill now, those who started their training with me, all had to go through the same basic steps. I will follow this method and we will write comprehensive protocolls about these steps.

The basic route in starting to work in our molecular biology lab was the following:

I. INTRODUCTORY PART

1. Introduction to the members of our lab.
2. Safety rules and regulations in the lab.
3. How to deal with the garbage, trash and other materials produced during the experiments.
4. How many tipes of water do we use?
5. What kind of reagents to we use?
6. Where and how do we store our reagents and our samples.

II. BASICS IN THE MOLECULAR BIOLOGY LAB

1. How can we protect our sample from degradation? If our sample is:
a. DNA
b. RNA
c Protein

2. DNA purification methods.
3. RNA purification methods.
4. Quallity control (QC) of DNA and RNA

III. BASIC TECHNIQUES TO GET USED TO LABWORK IN THE MOLECULAR BIOLOGY LAB

1. Working with plasimds:

a. Transformation
b. Growing of bacterial culture
c. Plasmid purification
d. Restriction analysis
e. Gel electrophoresis

2. Working in the cell culture lab:

a. Cell culturing basics
b. What is "sterile" in the issue culture environment?
c. Adherent and floating cells.
c. Making a passage.
d. Cell counting.

3. Transfection

a. Our methods used for transfection
b. QC
c. Sample preparation

4. PCR

a. Introduction into PCR
b. PCR and QPCR in practice
c. Analysis of the results

IV. ADVANCED TECHNIQUES IN THE MOLECULAR BIOLOGY LAB

We will elaborate this section later. We plan to include Western Blot, Protein purification, Modility Shift studies, Chromatin studies. etc.

Dear Colleagues!

I decided to make an online collection of the basic (and not so basic) techiques we use in our lab. This is a (hopefully) classical molecular biology lab located in Europe, Hungary, more close in Debrecen. I will present you the lab and environment later.

The idea is to describe these techiques, make a pdf version of the protocolls we are using and in some of the cases to upload videos about these techinques.

Any feed-back is wellcome.

Please stay tuned,

Balint