But is this the best way to do things? Should you allow yourself to succumb and be swept along in this frenzy in order to demonstrate your “flair”? Should you rush into your science as fast as your feet (or your brain) can take you? I would strongly argue that you shouldn’t. And here’s why.

Rushing means cutting corners. Cutting corners means skimping on the planning. And skimping on the planning can often equate to science that is flimsy at best.

Seasoned scientists can, if you slow down enough to ask them, give you close (and often terrifying) accounts of projects crashing down from their flimsy foundations. Those stories, sad and true, can be distilled and recycled into very simple and important advice.

Never. Rush. The beginning. Ever.

No matter how impatient your brand new boss is, how confident you (or he/she) is in the rapid success of your project-to-be, how fancy and readily available the resources of your new institution seem. Never rush the beginning.

Blitzkrieg science does not necessarily equate good science. In fact, it may represent quite the opposite.

Take a step back before you jump

Taking a step back to thoroughly assess the viability of your project (the many aspects of it) can seem like a waste of your precious bench time, and will not give you fast results to present in your first lab meeting.

But it might actually be the one thing that can steer you clear from failure. There is a lot left to luck even in the best planned scientific endeavor, so you are definitely better off keeping as much as you can reach under control.

A proper experimental design is the best, and in many cases only, way to guarantee a productive end to your research. Here are some hints to help you improve your project design, and therefore make you a better, happier, and undoubtedly more successful scientist.

1. Start your timer. Get honest with yourself about how long you plan to spend on your project, taking into account your program requirements, fellowship status, and your general life goals, if applicable.

Then look at the biological question you have chosen, and try to make a fair assessment of how long it might take you to come up with a valid, interesting answer.

Do you have to generate the necessary reagents and techniques? Do you have a working experimental model? Have you streamlined your data collection? How long will it take to obtain each sample? Do you have mid points or do you have to wait until the end to get meaningful data?

Typically, you might find yourself unable to readily address all those aspects, even using your best foreseeing abilities. What is relevant is actually asking the questions.

They will not only allow you to gauge the difficulty of the project, but it will also kick start your experimental design and guide you through the other important checkpoints.

2. Read critically. It always pays off to spend some time delimiting the boundaries of your project, by putting it in the context of previous data and existing literature.

Read extensively and leisurely about all aspects of your future work, technical and conceptual, and start out up to date with the current knowledge.

Chances are that you will have to struggle to keep up to speed with it from now on. The more information you gather the sooner and better you can focus your work into the most meaningful and interesting direction.

But while you are at it, develop a critical voice in your reading. Many research papers are told from the endpoint, making them sound like a seamless story in which all the experiments were conveniently and elegantly done in a neat chronological order.

Being critical with what you read will help you identify the failed approaches between the lines, so you can avoid them as well and save yourself time and chagrins. Find a poignant, clear spot to place yourself in science, and start building from there.

3. Look behind you. If you are lucky, you will discover that the foundations of your project have already been laid down by its previous “owner” in your lab.

In the midst of this initial excitement, do not to rush into the ritual of transferring the data and reagents from that person.

Firstly, take the time to ask as many question as you can think of before they move on. The more you are immersed in the literature, the easier that will be.

Both their data and reagents are going to be instrumental for your work, so take time as well to determine their quality.

Re-test the tools you inherit to your own satisfaction. Sequence your plasmids, blot your cell lines and checking the availability of stocks. This is of the utmost importance, and should override any concern you might have about good lab manners. Be a good scientist and everyone will treat you like a nice one!

4. Mind your “n”. Most projects stem from experimental designs based on statistically significant differences between control and experimental groups.

The statistical power of the tests that fit your design, and the depth and extend of the differences you expect to observe, are going to determine what sample number you will need to draw meaningful conclusions.

If your samples are wells on a tissue culture plate that you can re-seed day in and day out, you can easily increase the numbers should you end up requiring a bigger n, or if you want to add new experimental conditions down the line.

However, if you samples are lab animals that need to be trained for months, or specimens that need to be treated for long periods of time, or in any other way have an unfavorable ratio of time input vs. information output, you might find that increasing a sample number is not feasible once in mid-project.

To avoid that very frustrating pitfall, use the available preliminary data or similar studies from the literature to calculate or infer what you appropriate n might be. Then revise the backbone design of your project accordingly.

And as your rule of thumb, plan for the worst and give yourself some wiggle room. With honorable exceptions, there is not such a thing as too big an n.

5. Gear up. Before you get things rolling, figure out whether all the equipment that you are going to need to take your experiments to completion is available, and within reach.

Flow cytometry, microscopes for in vivo imaging, gradient machines, HPLCs, phosphoimaging hardware, deep sequencing infrastructure, animal facilities… whatever the requirements of your project are, make sure you will be able to access them when you need to.

That of course does not just mean addressing the physical presence of the equipment or resource.

Make sure to check the state of conservation, the use and maintenance policies and whether it is used frequently enough to assure that you will find someone to troubleshoot with.

Speaking of guidance, go ahead and start procuring some.

6. Get help. It is a widespread misconception that the best lab scientists are the ones that know almost everything and only ask very clever questions. But it is very unscientific  to approach an unknown territory without getting all the preliminary information about it that you can gather.

Because in the end, you will be judged based on what you have learned, not on what you already knew. So develop a good rapport with senior scientists around you, and never be afraid to ask for explanations, or to tag along.

The most stupid question can easily turn out to also be the most important one, and the best way to learn something is by watching an expert do it.

7. Prepare for the worst. The inherent risk of failure in your project cannot be eliminated within the parameters of scientific research, but it can definitely be lowered to almost harmless levels by means of a good design and some extra planning.

This can only be done at the initial stage, so one of your first tasks should be to stop and write down, next to your hypothesis, all that can go wrong and lead you to a negative, unpublishable result.

The ideal projects are those that are informative and make some advance regardless of the flavor of the result, but those are not easy to come by, especially for rookies.

So alternatively, try to revamp your yes-or-perish project by designing and implementing alternative strategies.

One easy way is to take on collaborations or side projects, that you can resort to if things go the “hypothesis 0” way. Bear in mind that handling many projects can in itself bring by disaster if you do not learn to prioritize as well.

Finding the right balance between too focused and too distracted will likely take some trial and error, and probably some ardent disagreements with your boss. Weight up your options, but remember that partial failure is still the opposite of a complete one.

8. Climb the chain of command. As much as you are bound to feel like you are alone in your project, you really are part of an intricate network of ideas, materials and discussions.

These are shared within, and expanding outside, the limits of your lab. Finding and defining who is involved in the project with you has two important purposes: giving other scientists proper credit and knowing who to report to.

Once you spot them, it is also helpful to figure out early the availability of your supervisors, so you can identify your daily go-to mentor and set up a discussion routine with the higher-up ones.

This will help you time and process your experiments to get the most productive feedback out of your mentorship meetings. Peer discussion and input are as necessary as a well-calibrated set of pipettes. So if you are still not convinced of the urgency and importance of this, please go back to #6 and keep reading.

9. Read this article. As many times as you need, and read many more like it. There are surely people out there tripping over the same (mile)stones, and learning the same hard lessons.

Get out and squeeze your library and computer dry for all the knowledge and advice you can get. And then have your pick. Be critical, experience for yourself, and give your advice back. Discover your own new solutions, and integrate them into the general wisdom pool.

It might not be officially recognized, but it is as good a way to contribute to the advancement of science.

10. Prove the principle. Once your experimental design has been thoroughly laid out, shake it and bake it. Repeating, or testing anew, the experiment that provides the structural and logical basis for your project is good science at its best.

Moreover, it will warm you up for the lab work to come. So if you know your way well enough around a lab, the best thing to assess the bench-readiness of your new stomping grounds is to actually go ahead and do some science.

When you are ready, embrace your project fully and just pour your excitement into it.

And learn to enjoy the discovery process, making it more meaningful than the unpredictable end result.

That is the ultimate way to ensure your personal success.

Now we have quick and easy gel extraction kits, we no longer need to use time consuming old fashioned methods like electro-elution or “freeze and squeeze”. Thank goodness.

When Gel Extraction Goes Wrong

But even the simplest of procedures can go wrong.

Maybe you were distracted, confused or thought that gel extraction was so easy that you tried to multi-task one too many things alongside it. (We’ve been there!)

However it happened, the good news is that if you make a mistake with these simple kits, it is still easy to fix.

A question from DJ…

We received the following question from one of our readers, DJ.

By mistake during my gel extraction I eluted with hot water (a tip I got from this site) before I had gotten rid of the buffer PE (primarily ethanol).

I get a decent amount of dna for my ligation purposes but the 260/230 is around 0.03-.1. I evaporated all the liquid in a vacuum centrifuge.

Then I resuspended in 70% ethanol to desalt it. I stuck it back in the vacuum centrifuge. When I resuspended it with water and nano-dropped it, the same 260/230 readings were returned to me.

What could I be doing wrong here that is not allowing salt to be evaporated from my DNA? Thanks! Love the site.

As Nick described in the early days of Bitesize Bio, a low 260/230 ratio is indicative of several possible contaminants.

EDTA, guanidine salts and oligosaccharides can all absorb around the 230 wavelength. The PE wash step is used to remove the left over gel and the salts from the column.

EDTA is usually not a component of wash buffers. But since the buffer formulations in these kits are proprietary, there is no way to know if a low level of EDTA is contained in PE.  EDTA is a nuclease inhibitor so adding it into buffers may be a way of ensuring DNase activity is eliminated.

When mistakes occur, it can be hard to troubleshoot when you don’t know the formulas for the solutions you are using.

But the chemistry is really simple (bind silica with chaotropic salts, wash away salts with ethanol, dry to remove the ethanol, elute in low salt buffer or water) and fixing slip ups does not have to mean starting over.

So what could have happened to your sample?

Since you eluted too early in the protocol, your sample would have contained guanidine salts, agarose and prehaps EDTA.

When you dried it down, you also concentrated those chemicals. With a 70% ethanol wash to remove residual salt, you may have removed some of it, but probably not all of it because when guanidine solidifies, it becomes difficult to resolubilize and usually needs some heating. So you did get rid of the ethanol, but probably not the guanidine.

If the contaminants were simply salts or EDTA, then ethanol precipitation would be a better way to clean up the DNA instead of drying it down.  If the contaminant was gel residue, ammonium acetate should be used for the precipitation salt because it will keep oligosaccharides soluble.

Forget the chemistry - just use a kit

If all of that chemistry is sounding like a bit too much hard work, you will be relieved to know that there is another solution that you could try if this happens again:

Simply re-bind the DNA to a new gel column by adding back the QG binding buffer or if you have a PCR clean up kit in your lab, you could use that to clean up the DNA on a new column. You should be able to recover 80% back (according to manufacturer data).

More gel extraction help…

You can get more tips on gel extraction (15 in total!) in a couple of articles, one  by myself and one by Nick.

But now that I am in gel extraction troubleshooting mode… here are a few more:

1. Always perform the additional QG wash to remove residual gel debris, especially if you pushed the limit of the max allowed for gel size, and then perform a second ethanol (PE) wash to make sure all the salt is removed.

You can perform a second wash with 80% ethanol instead of the PE wash so you don’t run out. Of course, dry the column before elution, but at least if you do forget, then the speed vac method will work to clean it up.

2. Only 100% anhydrous ethanol should be used in silica column wash buffers. Using denatured alcohol (typically the outer container will say 95% alcohol) will cause many problems with the drying step.

Denatured alcohol contains chemicals such as isopropanol or benzene that do not evaporate quickly and this carries over into the DNA.

You know when you used the wrong ethanol when your DNA smells funny or it won’t freeze in the -20C. And when you try and load it on a gel, it floats, even in loading dye.

The UltraClean Gel Spin Kit and UltraClean 15 DNA Purification Kit are examples of two gel extraction products that provide wash buffers already containing 100% ethanol  so you don’t need to add your own and mistakes like this can’t happen.

3. Remember your spec has a limited linear range!

If you are using a Nanodrop to quantify the DNA,  the readings tend to not accurately reflect the purity if the sample is near the lower level of detection for the instrument. I usually see this effect at around 10ng/ul and below.

When the DNA concentration is very low, the 260 reading is not peaking so the scan looks more like a straight line instead of a bell curve. But if the scan looks like a ski slope (starting out high at the 220-230 side and then sloping down as it gets to 260), then you do have some salt or other contamination in there. The scan will tell you more information, so the 260/230 may be fine and it may just be an effect of low yield.

If you are using a spectrophotometer, the same effect will apply. And, of course, for UV analysis of DNA, if you eluted the DNA in a buffer with EDTA, such as TE, then blank the instrument in TE.

4. For more tips on quantifying DNA with a spectrophotometer, check out this  Cold Spring Harbor Labs Protocol.

5. Go easy on the multi-tasking… you only have one pair of hands!

We’d like to know what you think of our gel extraction tips, or if you have any ideas or questions of your own. Tell us yours by leaving a note in the comments section below this article.

Non-fiction plods and trudges. However, ‘The Emperor Of Scent‘ by Chandler Burr is breathtakingly unique. It gallops. It has all the elements of a quintessential page turner. And it’s about science too.

It got me so electrified that I repeatedly found myself reading diagonally, reaching a point where I realised I’m not understanding anything because I’m going too fast, and taking a deep breath as I turned the pages back and re-read it.

The book is about Luca Turin’s (a biophysict for want for a better word, biophychemist to be exact) dramatic course of discovery of an alternative theory on how we smell.

I say ‘alternative’ because Wikipedia says so. But personally, I was thoroughly convinced that it is the theory on the mechanism of olfaction.

The theory is based on the bizarre idea that we smell using an electron spectroscope in our nose.

No, I’m not kidding. Yes, it is achievable using proteins. Yes, there are conclusive experiments for the same. No, he hasn’t yet got a Nobel. But you will be convinced that he deserves one when you turn the final page.

Isn’t it strange how we never give an iota of thought to how we smell?

Turin has a quirky, interesting character which absorbs one immediately. He’s passionate to the verge of obsessed. He’s restless. He’s intentionally oblivious to scientific protocol. He dabbles in everything, talks to everyone: Perfume, tracing submarines, electron tunneling, spectroscopy, insulin, transistors. He has thrillingly eloquent descriptions of scents.

And amazingly, it’s this rich soup of ideas, this intended scorning of the scientific boundaries (Physics, Chemistry, Biology), this compulsive desire to investigate just for the sake of investigating, that allows, and is absolutely essential for his theory to have been conceived.

The book also is scattered with all kinds of witticisms, concepts, and ideas that got my neurons tingling. I’m a sucker for scientific analogies. And Chandler Burr generates them as well as anyone.

He likens wave numbers to musical notes. He personifies electrons to explain electron tunneling in a way anyone can understand. Finally, the book also throws light on the myth we have about science and why it isn’t as perfect as we (or I especially) are wont to think of it.

I will have to read the book thrice at least, to enjoy all those tiny subtleties I missed while I stormed through it. I highly recommend it to anyone who loves science.

To conclude, some excerpts:

(Opening lines): Start with the deepest mystery of smell. No one knows how we do it.

(On Turin’s eloquence with scents): Read here

(On biological theories): Biological theories are created by pretending to be God. Another way of saying this is you put together a biological theory by reverse engineering the human body. You build a theory by looking at what already is and then try to think up a good reason why it would be that way, and how it would work.

(On Phy, Chem, Bio): It is said that both chemists and physicists study the atom, but chemists mess around with the electrons and physicists pass their time on the nucleus. Biology has now metamorphosed into the study of the gene….This is the historical reason people still say “molecular biology” which is actually a name without any meaning, As if there were any other kind of biology anymore. (This had me giggling for a while)

If you have read this book or are interested in Luca Turin’s work, please drop me a comment.

And/or if you have read a great book that you’d like to review on Bitesize Bio, please drop us a line!

The National Cancer Institute (NCI) asks for $50-$55 million worth of plans for biomarker research and $25-$30 million for cancer intervention and surveillance modeling.

The National Institute of Allergy and Infectious Diseases (NIAID) is asking for applications for $20 million to be available in FY 2010 for research on enterics and for investigation of agents relevant to biodefense.

The National Institute on Alcohol Abuse and Alcoholism (NIAAA) is looking for research plans in its programs for Core, Specialized, and Comprehensive Research centers, which would receive approximately $8-13 million in fiscal year 2011.

The National Institute of Child Health and Human Development (NICHD) seeks $8 million in 2010 proposals for research on rehabilitation and fertility preservation.

And to support career development, the National Heart, Lung, and Blood Institute (NHLBI) is offering $3.8 million for mentoring and diversity programs.

For more information, see the program announcements hyperlinked from the table:

I have 3 diplomas in Fashion Designing, an undergraduate degree in Medicine and a Masters in Business Administration with an emphasis in Marketing….I need some career advice…

This situation will be familiar to many of our readers. How many of us truly knew what we wanted to do when we were 17 or 18 and heading to college? We may have had an idea….but we didn’t really understand what those careers might entail.

Fast forward 8 years and you are well-educated and you have a ton of debt and are still trying to answer the question that plagued you at 18 – where do I go from here?

What do you want to do?

The first thing you need to do is to choose what you want to go. Which of those career directions do you want to take?

Only you can answer that and it may take some research and soul-searching to do so.

Browsing through current job listings of the sort you think you might be interested in and talking to people who are in those jobs will help focus your mind.

Once you have decided on your chosen path, then this becomes an exercise in
positioning yourself. And that means working hard on your resume.

Critical note: what you put on your resume is as important as what you leave off.

Your resume is a place for you to showcase why you might be a fit for a specific position….not just a general mish-mash of your background and everything you have ever done.

If you decide to pursue a position in fashion, I would suggest removing all references to your scientific experience, apart from the MBA is a great cross-over degree that applies to just about any position.

If you were focused on pursuing a position in science (since you are reading Bitesize Bio, my guess is that this is the case), remove all references to design.

Focus your entire background on scientific studies, courses, projects, experiences, etc. Keep the MBA of course – this is always a great degree for biotech professionals.They tend to put a lot of reverence in the MBA (something I personally never understood. As an MBA, I was always far more impressed with the MS and PhD folks….I guess the grass isn’t greener!).

Think about your resume the way you think about presenting a poster – do you put everything you ever did on the poster? Of course not (if you do, check out our 10 tips for designing research posters immediately for some guidance!)

You put the relevant data to communicate your findings…same goes for the resume.

If you can’t decide what you want to do…

…and I have a sneaking suspicion that this might be the case, your focus should be on getting a job, hopefully in an area that interests you.

In this effort you will be applying for a lot of different types of jobs e.g. in bench science, management or fashion. And for each of these, your resume should be tailored towards the requirements of the job.

So you should have a whole series of resumes.

Take these resumes and pursue careers in design, business and science until the right career path (i.e. job) emerges.

Remember this basic rule of job hunting:

It is always easier to find a job when you are employes. This applies to the fashion designer who wants to be research associate and to the marketing professional with a passion for design.

Whatever you pursue (whether it’s one career path or 3), be sure you only send information relevant to the position.

I hope this answers your question Vijay but if you have any questions, please put them in a comment below.

If anyone else has a specific career situation they need advice on, please feel free to contact us. We can’t promise to answer every question, but we will try…!

I’m assuming that you’re very familiar with how to identify relevant research articles using PubMed and Google Scholar. You go through the process of entering your search terms, getting a long list of results, scanning the titles, skimming an abstract, deciding you do indeed want to read the entire article, then jumping through some hoops, following a few links, and sacrificing your first born child to finally arrive at the full text article itself.

Well, Pubget takes those final steps out of the equation by not only helping you search for your articles of interest but also supplying you with the PDF—immediately—as part of the results, with no sacrifice of your offspring required.

SQUEE!!!

Okay, don’t be too excited, you’re not getting unlimited access to free articles. The only way to the full text is if it is open access or your institutional library has a subscription to the journal. You’re encouraged to add your institution if it’s not already listed.

The big deal, though, is that using Pubget offers a much much faster way of getting to the info you need. According to their Mission Statement:

Each year, scientists spend at least a quarter billion minutes searching for biomedical literature online. This is time they could better spend curing disease and building the future. Pubget’s mission is to give them that time back.

One really convenient feature of Pubget is that right on the home page it provides customizable links to top scientific journals, thereby allowing you to easily browse the most recent issues of your favorite publications and instantly read the PDFs of any interesting article. Again, it only works if you have access rights!

You can also create a personal library of “keepers” on the site, as well as take advantage of the Pubget RSS feed to receive automatic updates on your favorite topics without the hassle of recreating your search each time.

Right now, Pubget is free, although the developers mention that “premium services” are coming soon.

So check out Pubget for free while you can and see if it really does give you back your time!

“This effort will accelerate our understanding of how our bodies and microorganisms interact to influence health and disease,” said Kington in a
prepared statement. “Examining the differences between the microbiomes of healthy patients and those of patients suffering from a disease promises to change how we diagnose, treat and, ultimately, prevent many health conditions.”

The newly announced grants will support work at three centers, which will collaborate to sequence some 400
microbes of the human digestive tract, mouth, skin, nose, and vagina.

The centers are:

• Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX. (Richard Gibbs, PI; $3.7 million)
• Washington University Genome Sequencing Center, Saint Louis, MO. (George Weinstock, PI; $16.1 million)
• The J. Craig Venter Institute, Rockville, MD. (Robert L. Strausberg, PI; $8.8 million in 2009 American Recovery and Reinvestment Act economic stimulus funds.)

The Broad Institute (MIT/ Harvard, Cambridge, MA) “is expected to participate in this phase of the project.”

Here are the individual pilot projects announced today, listed by organ
system and project title:

• Skin: Psoriasis ($560,000). Martin J. Blaser, M.D., New York University School of Medicine,
  New York, NY.
• Vagina: Bacterial Vaginosis and Sexually Transmitted Diseases ($1 million).
  Gregory A. Buck, Virginia Commonwealth University, Richmond, VA.
• Male urethra: Puberty, Sexual Activity and Sexually Transmitted Diseases
  ($820,000).
  J. Dennis Fortenberry, Indiana University-Purdue University, Indianapolis, IN.
• Digestive tract, 2 grants: Obesity ($1 million) and Crohn’s Disease ($1 million).
  Claire M. Fraser-Liggett, University of Maryland School of Medicine, Baltimore,
  MD.
• Digestive tract: Crohn’s Disease
  ($980,000). Ellen Li, Washington University School of Medicine, Saint Louis, MO.
• Skin: Acne
  ($990,000). Huiying Li, University of California, Los Angeles, CA.
• Mouth and digestive tract: Esophageal Adenocarcinoma ($1 million).  Zhiheng Pei,  New York University School of Medicine,
  New York, NY.
• Vagina: Bacterial Vaginosis ($980,000).
  Jacques Ravel, University of Maryland School of Medicine, Baltimore, MD.
• Skin and nose: Atopic Dermatitis, Immunodeficiency Syndromes
  ($400,000). Julia Segre, National Human Genome Research Institute, Bethesda, MD.
• Nose, digestive tract and blood: Febrile Illness
  ($950,000).
  Gregory A. Storch, Washington University School of Medicine, Saint Louis, MO.
• Digestive tract: Necrotizing Enterocolitis ($1 million).
  Phillip I. Tarr, Washington University School of Medicine, Saint Louis, MO.
• Digestive tract: Pediatric Irritable Bowel Syndrome ($750,000).
  James Versalovic, Baylor College of Medicine, Houston, TX.
• Digestive tract, Crohn’s Disease
  ($1.1 million).
  Gary D. Wu, University of Pennsylvania School of Medicine, Philadelphia, PA.
• Digestive tract: Ulcerative Colitis
  ($1 million).
  Vincent B. Young, University of Michigan, Ann Arbor, MI.

Though there are no funding opportunities currently listed for the Human
Microbiome Project, as new grants become available they will be posted at

http://www.nihroadmap.nih.gov/hmp/grants.asp.

Additional information on the project is available at
www.nihroadmap.nih.gov/hmp/ and
www.hmpdacc.org.

I say obviously, but in my previous job as a technical services scientist, you’d be surprised at how often I found myself talking to customers about the importance of controls.

One customer commented, during our discussion over TA cloning problems, that a transformation control “…is just a waste of everything”

But in reality, the opposite is true. There is no simple way to know how good the cells are, than by doing a transformation control. And while an empty plate with no white colonies after an entire day of work is frustrating, having no idea what went wrong makes the experiment a complete waste.

So, where do you start with the controls?

1. Vector only ligation control: Often with TA cloning, people worry about vector ligating back on itself without an insert. If you are using blue-white screening, it is possible to see few white colonies with the vector only control.

However, it should not be more that 1-2% of the total number of colonies on the plate.

A vector only control (vector + ligase but no insert) gives you an idea of the kind of background you can expect to see on your experimental plate.

2. Transformation control: Every time a new vial of bacterial cells is removed from the freezer, it is good practice to perform a transformation control using a known amount of uncut vector.

The transformation control helps to show whether:

- The bacterial cells are competent
- Plates are fresh enough to allow growth of the bugs.
- You did everything right with regards to the transformation procedure itself.

3. Other Positive Control to consider: If you are really enthusiastic about having more controls, you could include a ligase control, using an insert and vector combination that worked in the past, to make sure that the ligase you are using is working well.

If your ligations are not going well, here are a few hints that might help:

1.Thaw the ligase buffer completely and mix well before using. White precipitates in the buffer could mean that one of the buffer components is precipitating out. 2 min incubation in 37C bath may help it go back into solution.

2.Multiple freeze thaws on the competent cells can impair their ability to stay competent. If possible aliquot them to volumes just enough for 2 transformations.

3.Prepare plate fresh if possible. Plates should not be stored more than a week at 4C. Do not add antibiotics when the liquid agar is hot. Warm the plates at 37C for at least 30 min before plating.

If you are only getting blue colonies on the plate…

Here are a few possible causes that most people do not consider:

1.Insert is too small to cause inactivation of the lacZ gene.

2.The insert is in-frame with the lacZ gene.

In the above two cases, you may see colonies that are light blue in comparison to some darker colonies. This just means that even though the cloning procedure worked, the insert was not the right size or of the right frame to result in a white colony. If the light blue colonies are screened, you may be lucky enough to get the clone.

3.The insert is toxic to the bacterial cell. Growing cells at 30C instead of 37C may help to some extent.

Hopefully these tips will help but if not you could take a look at our articles on pinpointing ligation problems (which provides some more details on ligation controls) and making competent E.coli (which includes a section on cell quality control).

Any questions or tips of your own? Drop me a comment!

To help you further in optimizing the RT process, here are some great enzymes and methods that can help you make a big difference in achieving high yields of difficult and low copy messages:

1. Choice of RT Primer

You have three choices of RT primer: oligo dT, random primers, or a gene specific primer. Many people use an OligodT primer so they can get full length copies of the mRNA.

However, if the message is long (>4kb) or does not have a poly A tail (prokaryotic mRNA), then you will want to use instead random primers. Random primers will enable you to transcribe 5′ ends of long genes, but your cDNAs may not be full length copies of the entire gene. Typically 6-mers are used but using 8 or 9-mers can help increase the sizes of cDNAs since they will hybridize less frequently.

For qPCR of eukaryotic genes, the approach of using a mix of long random primers and oligo-dT has been shown to give the best result (see Figure 23, page 32). Two companies that provide this type of optimized primer mix with their RT kit are Qiagen QuantiTect Rev. Transcription Kit and the BioRad iScript Kit.

The third choice is a gene specific primer. Gene Specific primers enhance sensitivity by directing all of the RT activity to a specific message instead of transcribing everything in the mix. If you are performing a one-step RT-PCR, gene specific primers are used because the RT primer is also your reverse primer for the PCR step.

2. Secondary Structure in RNA

RNA secondary structure can be a problem in transcribing full length RNA. The RT enzyme can stop or fall off the template when it hits loop structures in the RNA.

It is difficult to know if your RNA will have secondary structure, but typically if the GC content of the gene is high, it can be an indicator that the RNA is going to be difficult to melt apart and may not be completely single stranded before the reaction.For this reason, a usual first step is a 5 minute 65C denaturation to relax the RNA.

However, another approach is to use an RT enzyme that allows for synthesis to occur at a higher temperature than standard RT.   Thermoscript enzyme from Life Technologies is an example of a popular enzyme used for difficult templates.  Alternatively, there are RT enzymes with higher efficiency at moving through secondary structure, even at standard RT incubation temperatures (37C-42C), and have been shown to give better results with GC rich templates. An example of these are the Qiagen enzymes Omniscript and Sensiscript.

3. Removal of gDNA

Genomic DNA contamination in the RNA can be a cause of false positives in the final PCR. There are many ways to remove gDNA, such as DNase treatments during an RNA Prep or DNase treatments after a prep (such as the Turbo DNA-Free Kit by Ambion).

However, a better way to get around having to enzyme treat your precious RNA is to design primers that cross an intron or an intron-exon boundary. Using this method, no amplification can occur in DNA (or amplification of DNA results in a different sized band.) One problem to look out for is pseudogenes. A pseudogene is a DNA copy of the spliced mRNA inserted into the genome. Primers designed to RNA only will still amplify peudogenes.

For prokaryotic RNA which has no introns, genomic DNA removal becomes critical for accurate gene expression assays.  Enzymatic removal is the only choice and can be performed during the prep as described above, and for extra assurance that it has been removed, the Quantitect Rev. Transcription Kit has a gDNA wipeout buffer that removes any residual DNA before starting the RT and does not require heat or EDTA inactivation. This enzyme is recommended for qPCR because cDNAs may not be full length.

4. Check the RNA Integrity

RNA quality will have a big impact on the results of cDNA synthesis. And batch to batch variation in RNA quality will lead to inconsistent results. Before performing an RT, you should check the quality of the rRNA bands. One way to do this is to run an agarose gel and check it visually. Intact eukaryotic RNA should show a 28s and 18s rRNA with the larger band looking close to double in intensity compared to the smaller. If the intensity is about the same, it is still ok.

What you don’t want to see if heavy smearing around the bands and especially low in the lane. This is degradation.

If you don’t have enough sample to use on a check gel, a more accurate way of determining RNA quality is to use the Agilent BioAnalzyer. The BioAnalyzer provides you a visual representation of the RNA and calculates an RNA Integrity Number (RIN) to give you a quantitative measure of quality. A perfect score is a RIN of 10. RINs of 8 are ok. Below RINs of 7 and the RNA may have enough degradation to cause some problems detecting rare messages or providing consistent results.

They offer two different chips; one for detecting nanograms and one for picograms of RNA. This system is very expensive and so are the chips but if you have a core molecular biology lab or a microarray lab at your institution, they will likely have one of these instruments and can run samples, usually for a small fee.

For a fun and interactive presentation on RNA quality, check this out from Ambion.

5. Quantitating the RNA

Besides knowing the integrity of the RNA, accurate assessment of the yield is important as well. The accuracy of the yield can be affected by several things; accuracy of your measuring instrument, contamination with DNA, contamination with salts, and level of degradation. For measuring the yield, I prefer to use UV quantification using the Nanodrop.

This instrument does not require dilution of the sample and has a very wide range for measuring RNA. In our experience it can accurately read down to 10ng/ul. Conventional UV spectrophotometers with large cuvettes should be avoided for RNA because large volumes are needed for measuring samples. The downside of UV quantification is that genomic DNA will also be measured in the sample and if salts or phenol left over from the prep are contaminating the RNA, then there can also be added absorbance giving a false higher reading of the RNA.

Another way to measure RNA yield that gets around the problems of DNA and salt contamination is to use fluorescent dyes. Ribogreen is an RNA specific dye that can be used to measure yields using fluorescence. Nanodrop now has an instrument that can be used with Ribogreen.

6. Two-Step or One-Step RT-PCR

Whether to use a one-step or two-step RT-PCR is a question asked by all scientists. Shoba gave us some good advice on this topic for real-time PCR. There are advantages and disadvantages of each. With a one-step RT-PCR, a sample transfer step is eliminated, eliminating a potential source for contamination of controls. And with a one-step RT-PCR, because the primer is gene-specific, the sensitivity may be higher.

With two-step RT-PCR, a larger batch of RNA can be converted to cDNA and then aliquoted into many reactions. Two-step RT-PCR allows for greater choice in primers and also allows you to use the same batch of RT for analysis of multiple genes. This will help eliminate a potential contributor of variation when comparing different genes from the same source.

Regardless of which you choose, one-step or two-step RT-PCR, once you choose a method for your study, you need to stick with it. If you are performing qPCR, you will have a difficult time comparing data between the methods. If you just need a yes/no answer or are going to clone the cDNA you amplify, then switching back and forth won’t make a difference.

The reverse transcription process is influenced by many factors so to have the best results, it helps to design a strategy specific to each project. A method optimized for one project may not fit for another. It all depends on the type, level, and source of the RNA.

A thorough evaluation of strategies before you start will save you far more time in the long run.

Do you have any RT tips or questions you’d like to share…?

A: A huge, underscored, emphatic YES

Recruiters should have a prominent place in anyone’s career decisions.

They are, after all, professional networkers whose job it is to make the “match” between company and candidate. They know the lay of the land and can provide insight on what is happening.

Recruiters often have relationships with people you want to know and they have the time and the incentive to keep their networks thriving. These are people who can help you get your foot in the door, ensure the hiring manager actually reviews your CV, and help you navigate the treacherous waters of phone and face-to-face interviews…they’ve been there before and watched the people who made it and can help you do the same!

However, your career is an extremely important and sensitive topic.

Would I advocate you turning this crucial transition exclusively to a recruiter or recruiters?

Absolutely not. You are in charge of your career. No one cares about your career as much as you do. Recruiters can help you, but you have to guide them.

When you seek out recruiters, ask colleagues and friends for referrals. When you speak with recruiters, ask lots of questions.

• Don’t let them get away with giving you information that doesn’t make sense to you.
• Don’t let them send your information to a company without you knowing the name of the company!

You have to stay in control of the process.

But…. given proper guidance, a recruiter can super-size your career search.

They can get people on the phone that you might not be able to reach, they can leverage long-standing relationships and explain why you are a fit instead of relying on a resume.

Recruiters can be a tremendous advocate for you as these people will work hours and hours, making dozens of calls on your behalf in addition to all of the work you are doing – and they will not charge you a dime*.

* Run the other way and do not stop running if a recruiter is trying to charge you money to help with your career search. This is not standard practice within the industry and, to me, it is a horrid practice that preys on people unfamiliar with the industry.

Recruiters are not just for the active job seeker – and this is important.

Consider the economic events of the past 12 months and think about the people who suddenly found themselves out of work. Those with the best networks – including recruiters – were able to put together a formidable job search while the pink slip was still in their hands.

Even when you are working, take calls from recruiters occasionally, return their calls and if you find one you really like, provide them an occasional lead….they’ll love you for it!

Three quick tips on choosing a recruiter:

1. Reputation
2. Specialization
3. Gut Check (do you like them?).

In the end, these folks are there to help and most of them are genuine and helpful (those that aren’t…stop talking to them and go find someone else who you trust).

Have you had dealings, positive or negative, with recruiters? Let us know about them in the comments section.