Subscribe to Feed            Add to your Favourites

“It suddenly struck me that that tiny pea, pretty and blue, was the Earth. I put up my thumb and shut one eye, and my thumb blotted out the planet Earth. I didn't feel like a giant. I felt very, very small.” – Neil Armstrong (1930-2012)

Fresh Reads from the Science 'o sphere!

Wednesday, January 30, 2008

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.

**********

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.

6 Comments:

Lab Rat said...

I was going to make some snarky comment about molecular biology benchwork still being in the primitive, swirl-stuff-in-the-flask era, compared to what the nanofabrication folks do.

Then it occurred to me that biological dirt on multilayer thin film devices is merely a minor, easily-removable irritant, unlike biological dirt in biological samples, where presumably it becomes a major showstopper.

You have my deepest sympathies. :P

Lim Leng Hiong said...

To Lab Rat:

That's why it's called a "wet lab". Yes it is very wet indeed.

Wanderer said...

Hrmm...

Your blog has just been classified under "Pornography" by this web-filtering company called Fortiguard. I wonder why...

Lim Leng Hiong said...

Oh NOES!

JM Ridlon said...

I would also recommend designing nested primers if you are having problems with PCR. If you don't get a band you can also dilute the original 1/50 in DW and run the reaction again. I also want to recommend the clontech TITANIUM Taq PCR kit, which I have always had very good luck with templates below 2 kb. I also have a tip for cloning a PCR product. I always take my PCR product (2 ul) and ligate this into a pCR8 GW TOPO TA cloning vector (Invitrogen). Your polymerase always addes "T" "A" overhangs and this vector has these "sticky ends" and a covalently bound Topoisomerase enzyme which ligates the product. Then, if you have designed restriction sites into the primers, you can cut the insert, GENECLEAN purify the insert and ligate into a pre-digested expression vector. Really high efficiency in my experience. Hope this helps!

Lim Leng Hiong said...

To JM:

Thanks for the additional tips! Yes, in fact a fellow student just succeeded in getting a 4 kb band (long PCR!) using nested primers. And we do TA cloning routinely - I will be doing that soon for my set of constructs...