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 […]

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 […]

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 […]

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 […]

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? […]

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 […]

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 […]

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 […]

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 […]

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. […]