This is not a question I can answer in five minutes or less, or in between visitors to my poster at ASM. So I’d like to write a couple articles on this topic beginning with one on the skills to develop and acquire to make yourself attractive to an industry hiring manager.

I am glad that industry and  biotech R&D science is gaining in popularity among the new generation of scientists and they are actively asking the right questions to position themselves better for when the time comes to apply for jobs.  In this first article, I’d like to tell you more about the skills and qualities that will make you an asset to a biotech company. In a future article, I can tell you more about how I got my job and we can also talk about the different industry career paths and how to excel in biotech company culture.

1. Know how to search for patents. If you plan to go for an R&D position, then spend some time now learning how to find information in the patent and patent application databases. Focus on reading the claims. The claims of the patent tell you what part of the invention is protected and what is not.  Having an understanding of how patent claims are written to protect an invention will be impressive to an employer.  The next time you are using a “patented product” look up the patent number – it’s usually in the fine print of the manual. You’ll understand the product much better. At work, you will never be making legal decisions or interpreting the patent on your own. But you will be asked to read the patents to give your opinion and to find all the relevant intellectual property in an area and be able to discuss it with a group.

2. Don’t shy away from public speaking. Working in a biotech company requires you to be outgoing and to be comfortable speaking in front of groups. Those groups could be your team but more often it may be directors and vice presidents.  You may be presenting a data to the heads of a company, trying to convince them to spend money on your product idea. Or, you may be helping marketing by giving a seminar at a university or at scientific conferences. It is important not to shy away from these opportunities to be in the limelight. You want the heads of the company to know who you are and the quality of your work. So I recommend, if you are not doing a lot of public speaking now, such as department journal clubs and seminars, then get yourself into the rotation and make yourself do it. It just takes practice and scientists who can and do speak in public are promoted faster.

3. Collaborate with a local or a favorite biotech company. The very best way to get an idea of how biotech views science is to work with a company directly.  You can do this a number of ways.  You can offer to perform a side project that shows the capabilities of a company’s product and co-publish together.  If you have an idea for a product or a product improvement, you can see if the company will support you in developing it further. My company does this and we love it.  We don’t have the capacity to perform certain technologies in house, so by working with researchers we can generate data that would not be possible.  Here are two examples: one is a poster with T-RFLP analysis for metagenomic analysis and the other is a study on a new Soil RNA stabilization product using soils collected in Antarctica.  Neither of these projects would have been possible without the work by the collaborating lab. In the first poster, the scientists were undergraduates so this was especially rewarding. When they presented this work at ASM, people were incredibly impressed that this was done by undergraduate students. My lab did all the DNA preps and their lab did all the PCR and analysis (the hard part). It was a win-win situation.

Working with a biotech company on a project is a great way to understand the needs of biotech in science reporting and how to communicate science from a different perspective.

If you are too busy to take on a side project, then offer to manage a summer student or undergraduate student to partake in a biotech project with commercial value. We do this too, currently working with a group at the University of Maine that gave their ”Upward Bound” student a project using our products.

The benefits of doing this are many fold. The biotech company will often pay your way to a conference so you can present the work in a poster or talk.  Or they may even invite you to be a featured speaker at a large event. And with your data, they can publish “white papers” which are disseminated far and wide and can give your work more exposure to the community. We even publish these in Biotechniques in the “Advertorial” section.

And of course, a letter of recommendation from the director or managers of R&D at a biotech company will carry a lot of weight when you are going head to head with someone else without commercial science experience.

4. Get along with others. This might sound obvious, but, we all know that in life there are always people we do not get along with. In academics, we can usually ignore them since so much of research is independent and not collaborative. But in a company it is not the case. You will need to learn how to manage politics and to work with groups of people, some of whom do not get along with each other, and, for one reason or another, will not get along with you.  This is a difficult skill to develop and some people never do master it. Those people will not be promoted quickly in the company either.

My suggestion is to practice not holding grudges. As a postdoc, I worked for a PI that would always tell me “everyone is entitled to a bad day”, meaning, when another colleague was less than cordial or disagreeable, he would give them a pass. Basically, he said to let it go and assume the person is having a bad day and not take it personally.  Now, whenever I am treated in a way I don’t appreciate, I say the same thing to myself. Maybe that person is having a bad day. I’ll give them the benefit of the doubt.  It’s a good way to be and you’ll have a lot less mental stress if you can do it.

5. Don’t be afraid to go outside your comfort zone and try new things. In biotech, to solve a problem or to develop a certain technique, you may need to incorporate new assays that you’ve never done before. Embrace the opportunity to add a new method to your arsenal.  Sometimes it may be better to collaborate with a lab that has expertise in the method or to outsource to a company. But if you have the time and the money to learn something new and different, show your managers how fast you can adapt. Dealing with change is a big part of biotech science and those that handle change well tend to have an easier time in biotech and score higher in performance reviews.

In your academic life now, I would recommend that you learn many techniques, but not only for the reason of knowing the skill. For the reason that it teaches you confidence in yourself and your ability to learn anything at all.  It will help you to confront any new challenge with ease and make you a stronger scientist and a leader to your peers.

6. Be friends with your sales people. One of the best ways of actually landing a job in a biotech company is to know someone in the company who knows you and knows the kind of person you are. Most biotech companies pay their employees a bonus for referring people who are hired. So the employees of companies are always looking for new talent to bring in to their organization.  If you had the chance to collaborate with a company and do research for them, you’ll have an even easier time, since you’ll be dealing with marketing people and most likely the R&D people. They will already know how capable you are and how well you get along with others.

Some of you work at colleges or small schools that do not get sales reps visiting very often. In this case, just stay on good terms with the sales people who contact you through email and when you go to scientific conferences, be sure to visit the vendors. If you have a particularly favorite company, make sure to say hello and tell them how much you like their products. You can try to get the name of an R&D manager from the booth staff that you can talk to about setting up a collaboration.

Another really good way to stay in contact with companies is using socal media such as linkedin or twitter. Twitter is a great way to chat with individuals from your favorite company and give them positive feedback. Almost all of the biotech companies have a couple employees using twitter to chat with scientists and stay on top of current science events. If you join twitter, make sure to follow @bitesizebio and me at @suzyscientist.

7. Never, never burn any bridges. This is probably the single most important advice I can give you and that you will ever receive.  This is so critical to your career success and it is never too early to adopt this motto. This means, always leave every place of employment on good terms. In our world, you just never know when someone you were rude to will show up again. Even though academics and industry are very separate, so if you have a bad parting of ways with a PI, it won’t have as strong of a negative effect, you still want to make every attempt to smooth out any misunderstandings if you can.

Now, sometimes burning a bridge cannot be avoided. I know. I’ve done it.  But if I could do it over, I would have handled things differently. You might be thinking, “well you’ve done pretty good for yourself anyway.” Yes, but, it was rough going for a while. So save yourself the trouble now. If you leave a postdoc on bad terms, it will be difficult to get another postdoc or an academic position again. If you leave a company on bad terms, it could become very difficult to get hired again.

I know that sometimes it can be very tempting to want to lash out verbally when you feel someone mistreated you at a job. Don’t do it. And when interviewing for the next position, NEVER talk negatively about the lab you just left. I can guarantee that if you do, you won’t get hired.

Summary

As part of your training, you are going to learn a lot of techniques and you will develop your knowledge in your area of expertise. It doesn’t matter to me how many techniques you know as long as you know the ones I need you to know to start a project with the least amount of down time. Industry labs need people that can get up to speed quickly and not spend excessive time learning basic techniques. And we want people who are not afraid to learn and can get themselves trained quickly on new methods. Being self-sufficient is part of the Ph.D. process so being able to give examples of times where you learned a new difficult skill or technology on your own and quickly will make a very good impression to hiring managers.

As Greg Lucier said in his presentation on leadership,  it’s not only about what you know, but also how fast you can learn.

Divide by Three
Not only is the number of tubes a microcentrifuge can hold divisible by two, but almost always by three as well. How does this help you? If you have an odd number of tubes, you can now spin them without a balance tube. Here is the rotor from my microcentrifuge:

I have taken a red marker and colored the white numbers that divide the rotor into thirds, allowing me to quickly place three tubes into the microcentrifuge while maintaining balance in the rotor. On other microcentrifuges that did not have a paired position indicator (a white line on this rotor), I have used a different color marker to divide the rotor in half. Now what if you have five tubes?

The orange tubes balance each other, and the green tubes balance themselves. Of course you don’t need to break out the markers to do this, but premarking the rotor helps avoid mistakes when you are working quickly.

Nested Tubes
Some centrifuges come with multiple rotors that fit 2/1.5 ml, 0.5 ml, and 0.2 ml Eppendorf tubes, and it is best to use these when you are going to be spinning your samples hard for an extended period of time. However, changing rotors can be a pain and most of the time we aren’t trying to pellet our samples, but just trying to bring any condensation on the lid or the sides of the tubes down into the bottom. For these short spins, we can spin our smaller tubes in the 2/1.5 ml rotor by using some home-made adapters.

By cutting the top off a 1.5 ml Eppendorf tube, we can nest a 0.5ml tube inside it (left). And if we also cut the top off the 0.5 ml tube, a 0.2 tube can nest inside that as well (right). Depending on the particular tubes you are using, these nested configurations can tolerate quite a bit of force. However, if the samples are particularly precious, radioactive (or otherwise dangerous), or if you are going to be spinning them hard and long, you should probably go to the effort of changing the rotor rather than using these adapters.

Stock Balance Tubes
For those times when you just need a balance tube, you can save some time by preparing a selection of tubes with the volume written on the top and keeping them in a rack next to the microcentrifuge.  I know this seems really obvious, but most of the people I’ve worked with simply never thought to do this. I can almost always just grab one from this selection rather than having to make up a new one for each occasion. In addition, I find that it saves me from having the collection of unmarked tubes with clear, colorless liquid in them sitting next to the centrifuge that seems to disturb my lab safety officer so much.

If you have any other tips, let us know in the comments!

PCR amplification/cloning

One of the most common primer-based applications is cloning.  The desired amplicon and the vector restriction sites will largely dictate the composition of the primers.  A few tweaks, however, can increase the chance of cloning your gene on the first try:

-   Use a “GC clamp” at the 3’ end of each primer.  The G-C bond is stronger than the A-T bond, so a 3’ end enriched in G and C residues will bind with higher affinity.

-   Match the melting temperatures of paired primers.  If both primers have a melting temperature within 1-2 degrees of each other, it will be much simpler to optimize your PCR conditions.

-   Check for primer dimers or hairpins.  Any annealing between your primer pair or of one primer to itself will drastically affect the efficiency of your PCR reaction.

-   Add a few extra nucleotides upstream of restriction sites.  This will increase the efficiency of the restriction reaction (by giving the enzyme something to “hold on to”).  Since the extra bases do not anneal to the template, they do not need to be included in the melting temperature calculation.

Mutagenesis

Primer-based mutagenesis is a quick and easy way to mutate a gene of interest.  Mutagenesis kits contain extensive information on how to design optimal primers for mutagenesis, which can vary significantly from cloning primers in length, composition, and melting temperature.  Here are a few additional things to keep in mind:

-   Use the company’s Tm formula to calculate melting temperature.  The formula provided in the kit information will often yield a different melting temperature than that calculated by standard methods; be sure to use the formula suggested by the kit to get the right primer length and composition.

-   Try incorporating a restriction site.  If you can introduce a novel restriction site through a silent mutation, you can screen the mutated plasmids by digest.  This can save time and money while waiting for sequencing info on the putative mutated clones.

Websites and programs

Most sequence analysis programs include primer design features.  In addition, there a several fantastic primer design programs available on the web.  Here are a few of my favorites:

-   IDT OligoAnalyzer. This free online tool will analyze single primers or primer pairs for melting temperature, dimer formation, and hairpins.

-   Invitrogen OligoPerfect Designer. This online tool, which requires an Invitrogen account, will design primers for you based on target sequence.

-   Primer3.  A popular online tool which will design primers based on sequence or analyze your pre-designed primers by every conceivable parameter.

-   UCSC Genome Browser InSilico PCR. This website contains genome data for an extensive collection of research organisms, and will perform an in silico PCR reaction to confirm that your primers only amplify one product.

What are your tips for primer design?

The new Assistant Professor

The Good: A young Professor is almost certainly going to have a small lab, and in the first couple of years he or she is probably going to actually be in the lab, working beside you. They will be heavily invested in your success, since he or she is unlikely to be successful themselves if you flounder or fail. The amount of personal attention will likely be very high, and when snags are encountered, they will be tackled head-on, possibly by the Professor themselves. In addition, an Assistant Professor still has a strong sense of ‘lab-time’, the amount of time it actually takes to accomplish a particular experiment, keeping expectations reasonable. Since the Professor has likely spent several years laying out the foreseeable path of his or her research, the research goals and milestones are well defined. The research topic is going to be ‘hot’, since hiring committees aren’t likely to hire someone proposing to start their career on a well-worn path, and the Professor will probably be energetic, which can lend a sense of energy and excitement to the lab, making it a place you want to be even when your experiments aren’t going well.

The Bad: The young Professor likely came to where he or she is – the manager of a laboratory – without any formal or structured training in management. You are one of their first employees, and they are about to develop a management style by trial and error. Lucky you. To make matters worse, developing that management style probably isn’t their top priority. In addition, all that personal attention and investment in your success can add up to a lot of pressure, real or only perceived. Suddenly a grant deadline looms, and that assay you’ve been developing has to be working in two weeks. Many who work in these labs really like their advisor, and can start to feel responsible for the success (ie – tenure) of their mentor, which can be heavy responsibility to bear as a graduate student. In addition, the lab may go through some periods where funding is very tight. During these times, experiments may have to be designed around the price of the reagents (even more so than they normally are) and you may be required to serve as a teaching assistant beyond the normal requirements for your program, both of which could unnecessarily lengthen your graduate career.

The Ugly: Some young Professors only management experience is supervising undergraduates or technicians, and they might attempt to supervise graduate students the same way – with very little room for independence or independent growth. Some Assistant Professors move the paper train along by taking their students data and writing the papers themselves. In most career paths scientific communication is (arguably) even more important than the ability to design and execute a well controlled experiment, so while this practice is faster, it does a great disservice to the student. Some young Professors can be so distracted by their other duties, dismissive of the value of management, or just so naturally ungifted at management, that the lab becomes a completely dysfunctional environment before anything is done about it.

What to look for: You have little, if any, history to draw on here, so your information likely has to be first hand. When you rotated in the lab, did the Professor have a clear plan, or did he or she seem to be making it up as they went along? The later could be a concern. Did they maintain a professional distance in their interactions with you, or did they try to be ‘one of the guys’? It takes a very special person to effectively lead while still being ‘one of the guys’, and it is unlikely that a managerially-inexperienced Professor is going to pull it off. Before you join a lab, you can sit down with a prospective mentor and ask what their philosophy is on graduate training and manuscript preparation. If they don’t seem to have clear ideas or plans that they can articulate on the spot, then you should be concerned. Working in a young lab can be exciting and rewarding, but make sure you are being trained, and aren’t simply a tool that the Assistant Professor uses to gain tenure.

The seasoned Full Professor

The Good: This Professor has already passed through the chaos of establishing the lab and his or her own career. They probably have a larger lab with lots of equipment, senior graduate students, and postdocs – in other words, resources outside of themselves. Many have two or more grants, lending a certain level of economic security in the lab. They have a system for training graduate students and running a laboratory that has evolved over time to create a functional environment. They have publishing experience and a reputation that both contribute to getting manuscripts published in respected journals. If they are highly regarded in their field, then some of that reputation will rub off onto you and perhaps make finding a postdoc a little easier. What you are doing isn’t necessarily the key to the future of the lab, so you are more likely to be given the time and independence to develop the project and yourself. All in all, a very stable environment.

The Bad: While there are certainly many exceptions, sometimes all that stability comes at the expense of energy and excitement in the lab. The lab may be working on something that was cutting-edge 20 years ago when the lab was started, but may not be now. You may be working in a system that the field seems to have left behind, or working out the details of a process that was discovered one postdoc and two graduate students ago. These projects may be relatively safe, but unlikely to turn heads at a meeting and may not be motivating enough for intellectual adrenaline junkies. The independence you have can also have a dark side – time can start to stretch out while the Professor patiently waits for you to solve one technical hurdle after another. It isn’t uncommon for the lab management to have evolved via lowest common denominator (simply banning anything that somebody once complained about), leaving the lab heavily regimented with lab rules and a less than energetic and creative environment.

The Ugly: While senior Professors have a management style, it isn’t always a good one. Some tire of managing all the group dynamics in a lab, and start viewing their trainees as children whose behavior is simply an obstacle to research. Consequently, a very patronizing style develops that may actually hinder the development of professional behavior of the trainees. You can get lost in the crowd, not only of the current crop of lab members, but with those from the past. One Professor that I knew routinely called one of his graduate students by the wrong name – which he usually ‘apologized’ for by saying “Oh! Well, yes of course I know your name – you just remind me of him.” Some seem to give up any active role in mentoring at all, and view the tenure of the graduate student in his or her lab as simply an opportunity for the student to prove themselves, not unlike teaching someone to swim by tossing them into a lake and merely observing the results.

What to look for: The best way to determine if you will be happy in a well established lab is simply to talk to the current lab members. Ask how happy they are, and how they would characterize their interactions with their advisor. Ask about the general traits of people who have been successful and happy in the lab, as well as those who struggled. Then take an honest look at yourself and ask which group you’re most similar to. If there is a history of dysfunction in the lab and the current lab members aren’t happy, walk away. There is a real “But I’m special” syndrome that many first year graduate students contract that causes them to ignore history and join dysfunctional labs. In the end, it invariably turns out that they weren’t as special as they thought, and have all the same problems as the people before them did.

What I’ve painted here are the two opposite ends of the spectrum, and Associate Professors can fall anywhere in between. Some have the positives of both groups with few of the negatives, and sadly some seem to have only the negatives of both groups. In addition, all labs and Professors are different, so don’t make any assumptions based on a Professor’s academic age. (I’ve know Assistant Professors with two large grants working next door to a Full Professor struggling to get one grant funded; Assistant Professors that are natural leaders while some Full Professors that ‘mentor’ with curse-laden diatribes; et cetera, et cetera.) Gather all the information you can from your rotations keeping in mind some of the issues I’ve discussed above, then determine which environment fits you best.

If there are any other aspects of a new versus established Professor that a student should consider before joining their lab, please add them in the comments!

Ultracentrifuges generally come in two flavors: analytical and preparative. Analytical ultras are a specialized piece of equipment, and you can read more about them here. Preparative ultras are the simpler, more widespread machines that you are more likely to use in the course of purifying viruses, DNA, or proteins, and handle larger volumes that the analytical ultras.

These beasts of the lab spin samples fast enough to create centripetal forces as high as 1,000,000 times gravity. That is a serious amount of force, which deserves a serious amount of respect. Here’s what you need to know:

Balance your tubes
I know it’s obvious, but it has to be said. You CANNOT ‘balance’ your ultracentrifuge tubes by volume (ie – pipetting the same volume into two centrifuge tubes and assuming they’re balanced), or by sight (ie they look like they have the same volume in them). You need to put them on the scale and, as a rule of thumb, balance them to within 0.1 grams. If you’re using a tube with a screw-on top, then include the tops while weighing the tube just in case the tube/cap assemblies you are using aren’t perfectly matched.

Know Your Rotor
You might not think of centrifuge rotors as perishable, but they are. During the lifetime of a rotor, the metal can start to fatigue and form microscopic cracks. If you have a clumsy labmate that drops or bangs the rotors together, then these microscopic cracks develop even faster. Low speed rotors usually have an expiration date based on the manufacturer’s estimate of how often they are used. For precise estimates of a ultracentrifuge rotor’s lifetime, typically the rotor identification is recorded in a log book along with the speed and time spent running. (Newer ultracentrifuges keep track of this information in their on-board computer.) Based on this, the rotors are derated (the maximum rpm for that rotor is reduced) over time. So rather than knowing what the maximum speed for a rotor was when it was new, it is more important to know what it is now. Failure to do this could result in the overused rotor breaking during a run, undergoing what is called catastrophic failure. To be fair, however, almost any failure over 100,000g is catastrophic…

Know Your Tubes
The maximum speed of the rotor is only part of the equation. You also have to know the maximum speed for the tubes you are using given the volume of solution you will be placing inside it.  To put this a different way, reuseable ultracentrifuge tubes have a maximum volume and a minimum volume, and the maximum speed of the tube depends on the volume in the tube. A friend of mine unintentionally tested this idea several years ago, when they had to scale back a protocol, but tried to use the same centrifuge tubes they normally used in the protocol.  The result was a pair of collapsed tubes wedged into the rotor in a truly impressive manner.

In case you were wondering how many PhDs it would take to unscrew a collapsed ultra tube from a rotor, the answer is 6.5 – 6 PostDocs and a Grad Student.  (But 4.5 of the PhDs were ‘supervising’.)

The reason for this is that the fluids that we work with are, by and large, non-compressible; however, air is quite compressible.  So when the tube contains more fluid, it is more structurally sound.  So if your lab uses reusable tubes like the one above (it used to be reusable, anyway), make sure everybody using them knows the minimum and maximum volumes that can be spun in the tubes, the maximum speeds the tubes can be spun at those volumes, and the acceptable and unacceptable solvents and pH’s the tubes can be exposed to. There are some conditions that may weaken the tube without causing a noticeable effect on the tube.

If you are using thin-walled sealed tubes, the tube must be completely full or it will collapse.  Only open-top tubes in swing-bucket rotors can have air in them, but even these are usually quite full to achieve the maximum resolution of the gradients they often contain.

Pay Attention to the Vacuum Seals
The seals on tubes aren’t just there to keep the liquids in, but also to keep the vacuum out. Ultracentrifuges maintain low temperatures while spinning at such high speeds by running under high vacuum, and thereby reducing heat generated by air-rotor friction. However, if you exposed your sample to this vacuum, then it would start de-gassing your sample, potentially causing some liquid to escape even if you are using a swing-bucket rotor. To make matters worse, if you are spinning the samples overnight or longer, then exposure to the vacuum will cause that sample to evaporate while its paired sample doesn’t, potentially causing an imbalance in the rotor. So pay attention to the seals on the tubes or on the buckets for a swing-bucket rotor, they may require a little vacuum grease on the threads to seal properly depending on the design. For fixed angle rotors, there may be an additional seal between the lid and the rotor – make sure this is clean and undamaged before your run.

No Buckets Left Behind
One of the most common sources for ultra-disasters is somebody running a swing bucket rotor without all the buckets in place. These rotors are designed to be used with all the buckets in place, even if some of them are empty. Run a six-bucket rotor with only four buckets, and the rotor will deform and turn the four that are there into projectiles.  In addition, it is usually a good idea to have a small mirror handy to check that all the buckets are properly seated into the rotor once the rotor is positioned in the ultra.

So before you start using your laboratory’s ultracentrifuge, know that it is a potentially dangerous piece of equipment, but one that can be used safely.  Read the manuals, learn about your reusable and disposable ultracentrifuge tubes, and, if nobody else in the lab has, talk to the service technician for your ultracentrifuge to determine the maximum rate speeds for your rotors.

Do you approach the ultra with fear, respect or nonchalance? Have you ever had or witnessed an ultra disaster? Or do you have any other safety tips that I missed, please let us know in the comments!

Take 15 minutes to review the company’s web site. Review their history, what they sell, and key personnel. Be sure to look at other positions they have open - this will help you understand where they are focusing and provide some sense of their growth strategy. Take notes and devise a few questions that make it clear you did your homework.  For example, “I saw on your web site that (fill in the blank); how will that impact your department and the future of the organization?. These questions should not reference anything about benefits or salary!

Google the company – they may be googling you! A quick view of the top responses from this search engine may provide you with some interesting tidbits about the organization. This information is both good for you to know personally and to demonstrate your knowledge during an interview.

Make some notes about how your background ties in with the company’s background. Have you worked at similar companies in size and scope to this firm? Have you worked with similar products? Find a way to connect yourself with the organization while simultaneously demonstrating that you took the time to study them.

Be careful when asking questions that may show that you do not know much about the company.  For example, asking ”Who are your competitors?” can be risky in that it sounds like you didn’t take the time to find this information out for yourself. If you couldn’t find this information in your five-minute Google search, tell them. “I Googled your company, but was having a tough time identifying your competitors. Who is your top competitor?” Leverage your knowledge and prove your interest. This will help you set a strong foundation with the company and the interviewer.

What are your tips for preparing for an interview?

It isn’t often that one has the chance to receive career advice from the CEO of a billion dollar company in a small conference room setting such as this, so I took the opportunity to hear him speak.  Not only was it a great chance to learn some of his wisdom directly, but I also wanted to share it with you too here on Bitesize Bio.

Greg’s Background and Philosophies

Greg started off by telling the ~150 person audience about his educational background.  After getting a B.S. at Penn State, he attended Harvard where he received an MBA. Prior to taking the lead at Invitrogen, he was the General Manager at GE, working for Jack Welch, and learning the management style that worked so well at GE.  When he was interviewed by the Board of Directors for the position of CEO at Invitrogen in 2003, he told them ”hire me not for what I know, but for how fast I can learn.”

He had not worked in the biotech industry prior to this time, and except for the experience he gained in his evolving role at GE where he was managing their medical systems, life sciences would be completely new to him.  However, there was never a doubt in his mind in his ability to lead a completely different enterprise to greatness.  Indeed, when talking about key qualities in leaders, Greg stated, “The core of a great leader is self confidence.”

Leadership and Training

The bulk of the seminar was spent discussing how Life Technologies views leadership, and how they promote and train from within to move people with drive and motivation on to higher positions in the company.

When talking about picking the right players for his team, he was clear about what he looks for in a candidate for a leadership position at Life Technologies.  He said, “A person who has led an imperfect life.” A person who is reflective and can say “when they went left, when they should have gone right”, and talk about what they learned from their mistakes. Greg continued, “Look, you are going to [make mistakes] otherwise you’re not moving fast. You have to minimize it and the size of it.” And of course, learn from it. He stressed that people with battle scars make great leaders.

Creating a Great Workplace

Towards the end of the talk, Greg focused on the changes made at Life Technologies to make it a better place for employees, a place that people want to work. He spoke about how the GE method did not work at Life Tech and so, instead of asking people to change, he changed.  Now,  instead of ranking people from best to worst, they focus on people’s careers and individual growth and time balance.  He said they want employees to try different positions within the company, to learn new skills, and to further their education on the job instead of leaving to find greener pastures elsewhere.

As a former employee of Invitrogen (which merged with Applied Biosystems in 2009 to form Life Technologies), I can say it was nice to hear about the positive changes taking place at Life Technologies and how they are managing their rapid growth as a company to keep pace with the developments in science.

Greg’s perspectives on leadership were a great reminder that whatever it is in life that you lead, it is ok to try things and make mistakes, that change accelerates learning, and that self confidence can get you everywhere.

You can, of course, study the enzymes doing the phosphorylation/dephosphorylation, protein kinases or protein phosphatases, or the target proteins that are being phosphorylated or dephosphorylated. Each needs a different approach, so I’ll talk about both here…

Studying the Target protein

I personally like studying the protein targets of phosphorylation, since they’re the ones being phosphorylated, and by extension, the ones being regulated. If they’re being regulated, then, there must be some physiological significance as to why. So, what can we do to study phosphorylation of a target protein?

Enzyme kinetic parameters: If you’re lucky enough to have a target protein of interest that’s an enzyme with assayable catalytic activity, then you can see how that activity changes from a situation when you suspect your enzyme to be phosphorylated to a situation when you expect it to be dephosphorylated. Does the maximal rate of reaction (V_max) increase or decrease? How about the enzyme’s affinity for its substrates (K_m)? Does the activity versus substrate concentration profile shift at all- for instance, from a hyperbolic to a sigmoidal shape? Does the enzyme respond to the same activators and/or inhibitors in the same way? How does it function over a given pH range in its various phosphorylation states? The list goes on…

Detection by molecular imaging: There are also options for detecting phosphorylation on proteins that are completely independent of enzyme activity- in fact many variations of this next method preclude assaying for activity. Whether you use denaturing or non-denaturing methods, you can run your protein of interest- alone, or present in a complex mixture of proteins- out on a gel and try to detect it at the correct molecular weight. What can you use to detect it- a more appropriate question would be to ask what you can’t use! Nowadays, there are tons of commercially-available stains that are specific for phosphoproteins. Alternatively, for greater specificity and sensitivity, there are even lists of phospho-specific antibodies for probing your blotted membranes.

Mass Spec: This is essentially deserving of an entire post in itself, based on its diversity and complexity. Mass spectrometry, outfitted with a variety of accoutrements, is becoming an important tool in the characterizations of post-translational modifications on proteins, including protein phosphorylation. Phosphoprotein stains and antibodies can be a very useful tool in telling you whether your protein is phosphorylated, but not all of them will necessarily tell you where in the entire protein the phosphorylation is taking place- which site(s) in particular. By dissecting proteins in a mass spec, you have the ability to scan through peptide fragment after peptide fragment, scrutinizing which are different due to phosphates present on certain serines, threonines, tyrosines, and other phosphorylatable amino acids, throughout your protein’s structure.

Studying the kinases and phosphatases

Instead of – or, in addition to looking at the target protein, depending on the scope and objectives of your research and how keen you are- you can look at the protein kinases that are putting (a) phosphate group(s) on proteins, or the protein phosphatases taking it (them) off.

Kinases

Activity: Protein kinases are a world of fun in themselves, because right off the bat, they have the assayable catalytic activity to play with. The methods and tools for assaying kinase activity seem to multiply daily, and I myself have delved into only a few of the wide range available. You can measure activity using your protein kinase to phosphorylate a substrate radiometrically- using a radioisotope, ³²P-alpha-ATP, and transferring that hot terminal phosphate from ATP to the substrate. Here, again, what substrate are you using? A short peptide? A full-length protein? Will you spot it onto phosphocellulose paper, or precipitate it onto filter paper, or run it though acrylamide in SDS-PAGE? Alternatively, you can use methods that don’t even require those hazardous, inconvenient radioisotopes. I have used real-time assays that rely on the fluorescence of an unnatural amino acid, as well as endpoint assays that are based on the fluorescence polarization of an immobilized metal affinity peptide.

Kinases are targets, too! Anyone who knows about the introductory concepts of signal transduction knows that there isn’t just a signal kinase between a receptor and metabolic enzyme- there are thousands of protein kinases participating in extensive phosphorylation cascades. A kinase phosphorylates a second kinase, which in turn may or may not phosphorylate a third kinase, depending on whether its initial phosphorylation activated or inactivated it. And so on…

Phosphatases

A strange bunch! Whereas there are thousands of protein kinases, how many protein phosphatases are there? A few dozen, give or take? They seem to be much more promiscuous, in that they have broader substrate specificity. They are regulated by very different mechanisms, such as accessory proteins they associate with. Because they’re such an interesting niche, we’ll look at them more later.

Now that we’ve seen the candidates one can study when considering protein phosphorylation, next time, we’ll take a closer look at some of the tools that we only just skimmed over here.

These kits make the whole process much easier and faster than the methods of old, when things are going well, but the downside of using a kit is that we don’t always know what is in the mysterious and proprietary set of solutions that each company uses in its kit, which makes troubleshooting more difficult.

So in this article, I’ll explain in some detail  how silica spin filter kits work and what is going on at each step. I’ll also go over some common problems specific to silica columns that can be overcome or avoided with just a little extra understanding.

Lysis:

The lysis formulas may vary based on the whether you want DNA or RNA, but the common denominator is a lysis buffer containing a high concentration of chaotropic salt. Chaotropes destabilize hydrogen bonds, van der Waals forces,  and hydrophobic interactions. Proteins are destabilized, including nucleases, and the association of nucleic acids with water is disrupted setting up the conditions for the transfer to silica.

Chaotropic salts include guanidine HCL, guanidine thiocyanate, urea, and lithium perchlorate.

Besides the chaotropes, there is usually some detergents involved, to help with protein solubilization and lysis. There can also be enzymes used for lysis depending on the samples type. Proteinase K is one of these, and actually works very well in these denaturing buffers; the more denatured the protein, the better Proteinase K works. Lysozyme, however, does not work in the denaturing and so lysozyme treatment is usually done before adding the denaturing salts.

One comment about plasmid preps, the lysis is very different than extraction for RNA or genomic DNA because the plasmid has to be separated from the genomic DNA first and if you throw in chaotropes, you’ll release everything at once and won’t be able to differentially separate the small circular DNA from the high molecular weight chromosome.  So, in plasmid preps the chaotropes are not added until after lysis and the salts are used for binding. An excellent in-depth article on alkaline lysis is here and also another article on the difference between genomic DNA and plasmid is available for further reading.

Binding:

The chaotropic salts are critical for lysis, but also for binding, as we discussed. Additionally, to enhance and influence the binding of nucleic acids to silica, alcohol is also added. Most of the time this is ethanol but sometimes it may be isopropanol. The percent ethanol and the volume has big effects. Too much and you’ll bring in a lot of degraded nucleic acids and small species that will influence UV260 readings and throw off some of your yields.  Too little, and it may become difficult to wash away all of the salt from the membrane.

The important point here is that the ethanol influences binding and the amount added is optimized for whatever kit you are using. Modifying that step can help change what you recover so if you are having problems and want to troubleshoot recovery, that can be a step to evaluate further.

Another way to diagnose problems is to save the flow-through after binding and precipitate it to see if you can find the nucleic acids you are searching for. If you used an SDS-containing detergent in lysis, try using NaCl as a precipitant to avoid contamination of the DNA or RNA with detergent.

Washing Steps:

Your lysate was centrifuged through the silica membrane and now your DNA or RNA should be bound to the column and the impurities, protein and polysaccharides, should have passed through.  But, the membrane is still dirty with residual proteins and salt. If the sample was from plants, there will still be polysaccharides, maybe some pigments too, left on the membrane, or if the sample was blood, the membrane might be tinted brown or yellow.

The wash steps serve to remove these impurities.  There are typically two washes, although this can vary depending on the sample type. The first wash will often have a low amount of chaotropic salt to remove the protein and colored contaminants. This is always followed with an ethanol wash to remove the salts. If the prep is something that didn’t have a lot of protein to start, such as plasmid preps or PCR clean up, then only an ethanol wash is needed.

Removal of the chaotropic salts is crucial to getting high yields and purity DNA or RNA. Some kits will even wash the column with ethanol twice. If salt remains behind, the elution of nucleic acid is going to be poor, and the A230 reading will be high, resulting in low 260/230 ratios.

Dry Spin:

After the ethanol wash, most protocols have a centrifugation step to dry the column. This is to remove the ethanol and is essential for a clean eluant. When 10 mM Tris buffer or water is applied to the membrane for elution, the nucleic acids can become hydrated and will release from the membrane. If the column still has ethanol on it, then the nucleic acids cannot be fully rehydrated.

Skipping the drying step results in ethanol contamination and low yields. I do not see ethanol absorbance on the Nanodrop, so it won’t show up in your readings. The main indicators of a problem are that when you try to load the sample onto an agarose gel, the DNA will not sink. Even in the presence of loading dye.  Another indicator is that if you put the sample in the -20C, it doesn’t freeze.

Elution:

The final step is the release of pure DNA or RNA from the silica. For DNA preps, 10 mM Tris at a pH between 8-9 is typically used. DNA is more stable at a slightly basic pH and will dissolve faster in a buffer. This is true even for DNA pellets. Water tends to have a low pH, as low as 4-5 and high molecular weight DNA may not completely rehydrate in the short time used for elution. Elution of DNA can be maximized by allowing the buffer to sit in the membrane for a few minutes before centrifugation.

RNA, on the other hand, is fine at a slightly acidic pH and so water is the preferred diluent. RNA dissolves readily in water.

What other things can go wrong:

Low yields: If you experience yields lower than you expected for a sample, there are many factors to think about. Usually it is a lysis problem. Incomplete lysis is a major cause of low yields. It could also be caused by incorrect binding conditions.  Make sure to use fresh high quality ethanol (100% 200 proof) to dilute buffers or for adding to the binding step. Low quality ethanol or old stocks may have taken on water and not be the correct concentration. If the wash buffer is not made correctly, you may be washing off your DNA or RNA.

Low Purity: If the sample is contaminated with protein (low 260/280) then maybe you started with too much sample and the protein was not completely removed or dissolved.  If the sample has poor 260/230 ratio the issue is usually salt from the bind or the wash buffer. Make sure that the highest quality ethanol was used to prepare wash buffers and if the problem continues, give the colun an additional wash.

Some samples have a lot more inhibitors compared to others. Environmental samples are especially prone to purity issues because humic substances are solubilized during extraction. Humics behave similarly to DNA and are difficult to remove from the silica column. For this type of sample, specialized techniques exist to remove the protein and humics prior to the column step.

Degradation: This is more of a concern for RNA preps and an article that gives specific advice is here.  Mainly with RNA, degradation occurs from inproper storage of the sample or an inefficient lysis, assuming of course that you eluted with RNase-free water. For DNA preps, degradation is not a huge problem because for PCR, the DNA can be sheared and it works fine. But if you were hoping to not have so much sheared DNA, then you may have used too strong a lysis method.

PCR Clean-up Special Considerations: The easiest of all the techniques, because it is simply adding a high concentration of binding salts (typically between 3-5 volumes of salt per volumes of PCR reaction) and centrifugation through the column. So when PCR Clean-up kits fail, it can be particularly frustrating.  The first question I ask people is “did you check the results of the PCR on a gel?” because you cannot UV check a PCR reaction and have an accurate reading. There is way too much in a PCR reaction absorbing UV at 260: nucleotides, detergents, salts, and primers. In my experience, a failure of a PCR clean-up kit to work frequently is caused by a PCR reaction that has failed and so there was nothing to clean up. But if you know you had a strong PCR product, the best approach is to just save your flow-through fraction after binding. If the DNA doesn’t bind, that’s where it is. You can always rescue it and then clean it up again. And then call tech support and ask for a replacement kit.

Summary:

As scientists, of course we want to know exactly what is going on with our experiments and be able to troubleshoot without having to call technical service first. I hope that this article helps clarify some of the science around the common silica spin filter prep so you can make your own diagnosis.  So, when you do call technical service, you’ll have double checked a few of the most likely causes of problems first and instead of going through a lot of rigmarole, you can get to a resolution much faster. Even if that is a free replacement kit.

Any questions? Any other problems with silica spin filter preps that you don’t understand? Let us know or ask a question in our “Questions” section and we’ll discuss!

You’ve talked about your career, your ambitions, your salary requirements and your future. You love them and they love you.

Casually, they ask you for references - the final step in the selection process. You’ve come this far and now it is crucial you take the final step with the same level of professionalism and confidence that brought you here. But can you? Do you have the right references? What will they say about you? Are you positive that is what they will say about you?

It is important to handle references appropriately. Here are a few tips to keep in mind:

1. Contact your most recent direct supervisor(s) and ask to use them as a reference. Your new employer will most likely insist on talking to a recent supervisor before all other people.
2. Ask them what they see as your greatest strength(s) and greatest weakness. It’s better that you find out what they will say now . Alternatively, you can ask for a letter of recommendation. This will give you a sense of how passionately they speak of your strengths, though it will not provide insight on weaknesses they may discuss.  Letters of recommendation are not taken as seriously as phone references since your new employer knows you are a filter between your old employer and them and people tend to be less candid about their concerns in a letter format.
3. Once you have provided your references to a company, let your references know that they may be contacted soon. Ask them to please return the call promptly - a lot of references do not return calls for extended periods of time, and this can ruin the sense of urgency that your future company may have to bring you on board.

Keep these things in mind when responding to a company’s request for references:

1. Provide phone numbers and email addresses to your two or three most recent direct supervisors. Think about which references will be most relevant to this potential employer and which references will speak to your strengths. Companies find it peculiar when people list references from several years back and do not provide the most recent contacts.
2. Unless specifically requested, do not include personal or peer references.

If you follow these items, you can ensure the final step goes smoothly and increase your chances on getting a job offer.