Easy (all purpose) brain protein extraction

If you just want a positive control for your western blot and you need rat or mouse brain, there’s no need for a fancy and long brain protein extraction.  

- Dissect the animal and extract the brain

-  Take the whole brain or cut a piece of it (the region you are interested in) and homogenize it in lysis buffer and protease inhibitors (use at least 5 times the volume of the piece of brain)

- Centrifuge 10 minutes at 14000rpm at 4C

- Take the supernatant and dilute it in sample buffer or keep it at -80C to use later on.

 Your lysis buffer can be as simple as PBS (without calcium and magnesium) + 1% TX100 + protease inhibitors.

Buccal cell DNA preps

Important: Extract the DNA within one week of receiving samples. Samples that have been processed should be frozen to prevent degradation. Freeze samples between use each day

1. Add 600 ul of 50 mM NaOH to a 1.5 ml eppendorf tube and insert brush in tube. (Clip off handle of brush with wire cutters so tube can be closed. Cutters should be rinsed with EtOH between samples to prevent contamination.)
2. Vortex thoroughly to mix, for at least 10 s.
3.  Heat to 95C for 5 min.
4.  Spin briefly to pool condensation.
5.   Remove brush with tweezers (tweezers should be rinsed with ethanol between samples to prevent contamination) and add 60 ul 1M Tris pH 8.0 to tube. Vortex to mix for at least 10 s.
6. Spin for 1 minute at 13,000 rpm and decant supernatant to a clean storage tube.
7. Use 2-5 ul of supernatant for standard PCR.

From Chromaffin Cell & Hypertension Research Department at UCSD

DNA gel extraction trick

To cut the band in an agarose gel for dna extraccion, you need to visualize it first under UV light. And maybe you want to take a picture too.

Only a few seconds of UV light can damage and create new undesirable mutations in your dna. To avoid that and still have the picture of your gel, first cut the band as fast as you can under the UV light. Place the piece of gel where your band is in an eppendorf tube and then take a picture of the rest of the gel. Instead of your band there will be a hole, but still you will be able to tell where the band was, and keep it in your notebook for your records.

Another way of doing so is to forget about ethidium bromide and use a crystal violet agarose gel. But that’s a different story.

Don’t throw the membranes away!

Did you know that you can let western nitrocellulose and PVDF membranes dry up and keep them like this for a long time? Yes, you can ;)

I was doing that in my lab in Spain and when I came to US, I realized a lot of people was not doing it. When you are done with the western blot just wash the membrane with distilled water thoroughly to remove all ECL and put it on a tissue paper on your bench. Let it dry overnight and next day just put it in an envelope with the date and keep it in your notebook.

If you wanna use it later on, just add the blocking solution (or re-activate first with methanol in case of PVDF membranes) and it will re-hydrate. From here, proceed as usual. The proteins will still be there!

Actually, a friend of mine told me the other day that she has used membranes that she kept for as long as two years. And they worked!

So, you know now, recycling is good. You never know when you’re gonna need those samples again. Keep them just in case.

Methylation sites

Are you wondering why this enzime you are using doesn’t cut your plasmid? It happened to me! I was doing some easy cloning and there was no way I could get my plasmid cut. I tried several times, I bought a new restriction enzime… until I realized that my enzime was methylation sensitive.

What’s that? There is a very good explanation in the New England Biolabs website:

DNA methyltransferases (MTases), which transfer a methyl group from S-adenosylmethionine to either adenine or cytosine residues, are found in a wide variety of prokaryotes and eukaryotes. Methylation needs to be considered when digesting DNA with restriction endonucleases because cleavage can be blocked or impaired when a particular base in the recognition site is methylated.

In prokaryotes, MTases have most often been identified as elements of restriction/modification systems that act to protect host DNA from cleavage by the corresponding restriction endonuclease. Most laboratory strains of E. coli contain three site-specific DNA methylases. The methylase encoded by the dam gene (Dam methylase) transfers a methyl group to the N6 position of the adenine residues in the sequence GATC (1,2). The Dcm methylase, encoded by the dcm gene, methylates the internal cytosine residues in the sequences CCAGG and CCTGG (1,3) at the C5 position. The EcoKI methylase, M. EcoKI, modifies adenine residues in the sequences AAC(N6)GTGC and GCAC(N6)GTT. Some or all of the sites for a restriction endonuclease may be resistant to cleavage when isolated from strains expressing the Dam or Dcm methylases if the methylase recognition site overlaps the endonuclease recognition site. For example, plasmid DNA isolated from Dam+ E. coli is completely resistant to cleavage by MboI, which cleaves at GATC sites.

Not all DNA isolated from E. coli is methylated to the same extent. While pBR322 DNA is fully modified (and is therefore completely resistant to Mbo I digestion), only about 50% of λ DNA Dam sites are methylated, presumably because the methylase does not have the opportunity to methylate the DNA fully before it is packaged into the phage head. As a result, enzymes blocked by Dam or Dcm modification will yield partial digestion patterns with l DNA.

CpG MTases, found in higher eukaryotes (e.g., Dnmt1), transfer a methyl group to the C5 position of cytosine residues. Patterns of CpG methylation are heritable, tissue specific, and correlate with gene expression. Consequently CpG methylation has been postulated to play a role in differentiation and gene expression (4). The effects of CpG methylation are mainly of concern when digesting eukaryotic genomic DNA. It should be noted that CpG methylation patterns are not retained once the DNA is cloned into a bacterial host.

Also, if you go to the NEB web site, there’s a list of all the methylation sensitive enzimes.

What do you have to do to solve a methylation problem? In my opinion, the fastest way is to transform your plasmid in a dam and dcm negative bacteria strain, so it won’t get methylated. There are several strains of this characteristics: SCS110 or JM110 (Stratagene) for example.

One of the most used plasmids, pEGFP, has a methylation sensitive site. You will not be able to cut this plasmid with XbaI unless you transform it first in bacteria that generates unmethylated dna.  Hope this helps!

Good primer design tips

1. Primers should be 17-24 bases in length

2. Base composition should be 45-65% (G+C)

3. Primers should end (3′) in a G or C, or CG or GC: this prevents “breathing” of ends and increases efficiency of priming

4. Tms between 55-75ºC are preferred

5. Primer self-complementarity (ability to form 2º structures such as hairpins or primer dimers) should be avoided

6. It is especially important that the 3′-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesized preferentially to any other product

7. Simple sequences such as a run of 4 of any nucleotide should be avoided (eg. AAAA)

8. Runs of three or more Cs or Gs at the 3′-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.

Everything about western blots

Everything you always wanted to learn about western blots but were too afraid to ask is in this web site: http://www.westernblotting.org/

Western blot protocols, troubleshoting, definitions, buffers and theory all in a web site. What is biotin? how the stripping buffer works? how do I prepare the lysis buffer? In a very basic way, you’ll find your answers in this site.

Western blot troubleshooting

Here some problems we all have had when doing western blots:

- high background signal
- strange/non specific bands on the blot
- low sensitivity
- is a right band detected?
- uneven results with lots of spots

In the protocols section of the company Agrisera there is a good troubleshooting section that will answer all your questions. Visit it here: http://www.agrisera.se/protocols/westerntrouble.shtml

Also, you can keep the link to take a look at the rest of the page later on, as it is pretty useful and everything it is good explained.

Western blot protocol

A complete western blot protocol from Howel lab in University of California San Diego. Here is the link:

http://cancer.ucsd.edu/howelllab/Western.html

From how to obtain a cell lysate to how to prepare the gels, running the gels, membrane transfer, detection and stripping the membrane. Also all the reagents and buffers you need for your western blot.

There is many techniques, this is only the way they do it in their lab. You always need to optimize according to your protein of interest.

Cell trypsinisation

1. Remove medium from culture dish.
2. Gently wash cells in a PBS without Ca or Mg.
3. Remove the wash solution.
4. Add trypsin-EDTA solution to cover the bottom of the culture dish (2ml for 10cm dish, 0.5ml for 60mm dish).
5. Put dish in the 37°C incubator for up to 5 minutes.
6. Check cells under microscope. Cells are beginning to detach when they appear rounded.
7. When cells are in suspension, add growth medium supplemented with serum.
8. Dilute 1:2 up to 1:10.