Molecular Biology Tips

I'm really tied down with labwork these days, so here are some quick tips fresh off the bench. As usual these are my personal opinions only, but I'm quite sure they work:

1. PCR

A blank agarose gel is a depressing (and routine) sight in biological research.

What do you do when you don't get a band?

Newbies in biology may be too familiar with the myriad methods for optimizing your PCR reaction - MgCl2 concentration, annealing temperature, number of cycles, extension time... and so on.

But unless you really enjoy spending time and effort stuck in troubleshooting hell, here's a faster way.

I call it the "50 degree" rule of thumb.

If you don't get any bands, the first thing to do is to drop the annealing temperature way down and increase the number of cycles to an extreme level, for example to 50 degrees and 40 cycles.

You should get a band now. If the desired PCR product is short (<1000 bp) just do a bunch of PCR reactions and send them off for sequence verification. Pick the one with the correct sequence and move on.

If the PCR product is long, it's more important to optimize the annealing temperature back up.

If you still don't get any bands, then it's likely a problem with the primer design.

If it's for PCR screening, order a bunch of different primer pairs by "shifting" the primer position back and forth a few base pairs.

For subcloning work you may not be able to move the primer position. Order primers of different lengths instead.

Wasting primers is much cheaper than wasting your time.

2. Ligation

Ligation is one of the black arts of molecular biology. Sometimes it works right the first time, sometimes it doesn't ever work.

Yes, we know about the 1:3 vector-to-insert molar ratio calculation that you learnt in school. But there's no guarantee that the ligation will be successful.

I prefer a brute force technique - by using an overwhelming amount of insert DNA.

First, set the amount of vector DNA fixed at 100 ng.

Then add as much insert DNA as it can possibly fit in the ligation mix. (In a 10ul reaction you can add up to 6ul of insert which is 1000 ng or more!)

I find that adding 1 ul of 100mM ATP helps too.

3. Plasmid DNA extraction

You spent a whole day to extract plasmid DNA from a huge flask of bacteria. You finally get a tiny, shiny pellet in isopropanol.

Then, during the last purification step in 70% ethanol, you lose the pellet.

Oh the horrors!

Don't risk it. When doing a Maxi/Midiprep, if the pellet is already tiny (<2mm diameter) by the isopropanol step, it will become practically invisible at the 70% ethanol step.

In addition, it will become fragmented, flaky, loose and easily lost when you are trying to remove the ethanol around it.

In such a situation, I prefer to skip the additional centrifugation step. After removing isopropanol, simply rinse the pellet with 70% ethanol, suck away the ethanol and then dissolve the pellet in water/elution buffer.

The yield of DNA is high, the quality (260/280 ratio) is good, and I've used it for downstream subcloning work and sequencing with no problems at all.

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That's it for now - back to work I go.

Here at Fresh Brainz, we cry at the bench so you won't have to... cry as much.

Lab Safety Tips

Most labs require new staff and attachment students to attend basic safety training before starting work, so they are usually instilled with a fairly good safety sense right from day one.

However, not all safety issues are covered by the training. Some of these are highly specific to the nature of the task - for example biohazards and radioactivity.

Others are generally applicable in most research settings. Fresh Brainz would like to draw your attention to a few safety tips that will help safeguard against less obvious dangers in the lab (Note: These are my personal opinions only!) -

1. Gloves Are Not Invincible

An obvious way to know whether a person is a real researcher or just somebody who plays one on TV is to watch how she handles gloves.

There are two main reasons for wearing gloves - to protect the sample against contamination, or to protect your hands from hazardous materials.

In the first case, the gloves should be kept clean and sterile using regular sprays of ethanol. One should only handle the relevant samples with your gloves. Pay attention to where your hands are going. You should not scratch your face or receive a phone call while wearing gloves. When in doubt, change gloves often to avoid cross-contamination.

In the second scenario, it must be emphasized that gloves are protective but not invincible. If corrosive chemicals spill onto the gloves, they will slowly permeate through it. Thus, you should discard your contaminated gloves and replace them with fresh ones promptly.

There is a method for removing contaminated gloves safely. First, use a gloved hand to slide off a contaminated glove and drop it into the appropriate waste container. Next, slide a bare finger into the inside of the other glove and quickly turn it inside out while dropping it into the waste container.

Never use your bare hands to contact the outside of a contaminated glove. That utterly defeats the purpose of wearing gloves.

2. Keep Your Hazards Low

To reduce the impact of accidental spills, keep only small quantities of hazardous material enough for your protocol at the bench.

In addition, handle these reagents as close to the work surface as possible. If you keep them low, in the case of an accident the spills will usually be in a small area and easily decontaminated.

If you handle them high above the work surface, in the case of an accident the container will drop onto the bench/floor, splashing everywhere and causing a big mess that can be a pain in the ass to clean up.

3. A Sealed Container Is A Potential Bomb

New lab personnel are instinctively careful about chemicals and flammables. They are usually cautious about mixing chemicals and working with flammables, because they know that these can cause explosions if not handled properly.

However, it should be emphasized this does not apply only to reactive chemicals - any sealed container can become an explosive risk.

All you need to do is to add heat.

Thus, it is a good practice to check that containers to be heated in microwaves or hot plates have a way to vent their expanding gases.

A loosened cap on a glass bottle or flask is usually not enough, because the rapid gas expansion inside can set the cap tightly against the screw threads, blocking the escape of hot gases. A better method is to plug the opening loosely with soft material that can pop out harmlessly.

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Finally, here's another tip for non-lab personnel who are visiting research labs as part of an guided tour or "open day" exhibition.

Unless instructed, avoid touching anything in the lab with your bare hands.

In particular, stay away from the sinks, which may have traces of bleach that can ruin your clothing. A long time ago, an admin personnel had her expensive blouse damaged by a few drops of bleach when she leaned against a lab sink.

And after your visit, don't forget to wash your hands before going home - you wouldn't want to bring a Crankémon home to your kids!

Heh.