Month: October 2018

Preparing and running slide gels

Preparing slide gels

Slide agarose gels can be run in 7 minutes and give great resolution of DNA samples. Multiple gels can be prepared at once and saved for up to a month. Slide gels use only 0.12 g of agarose per gel which reduces the cost of running gels. See the accompanying video “Running slide gels” below.

Gel recipe: 10 mL 1x TAE buffer, 0.12 g agarose –> boil by microwave until dissolved (about 45 sec on our microwave) —> add 0.5 µL 10 mg/mL ethidium bromide

Running slide gels

Slide agarose gels can be run in 7 minutes and give great resolution of DNA samples. Multiple gels can be prepared at once and saved for up to a month. Slide gels use only 0.12 g of agarose per gel which reduces the cost of running gels. See “Preparing slide gels” above for casting the gels.

Super cheap plasmid miniprep without columns

Short version (Recommended).
Long version with more tips and details but poor sound quality.

This video protocol shows you how to perform an alkaline lysis plasmid miniprep. The DNA purification industry would like you to believe that their kits are necessary to obtain plasmid DNA suitable for routine applications. This is simply not true. In fact, isolating large quantities of high purity plasmid DNA is really easy.  Plasmid miniprep procedures commonly use columns that give low yield and are relatively expensive. This modified version of the traditional alkaline lysis miniprep produces large yields of high quality DNA and costs less than $0.05 per prep (including cost of tubes). Download a printable plasmid miniprep protocol here. The protocol described here (and shown in the video) has a few modification from the classical miniprep protocol found in books such as “Molecular Cloning” that increase the purity of the plasmid DNA.

Advantages of this miniprep method

  1. Large yield
  2. High purity
  3. Fast
  4. Cheap, 20 times less than most commercial kits.

Tips

  • The hole in the cap of the tube can be taped shut after cultures have finished growing, when using 2 mL microcentrifuge tubes to grow cultures. Pelleted E. coli can be frozen at -20 degrees C after the supernatant is discarded and processed later.
  • It is important not to transfer and white precipitate to the new tube at step 8 in the protocol. However, you will have a second chance to get rid of the white precipitate later. When resuspending the ethanol pellet in Tris buffer (step 13) you may have some white precipitate that does not resuspend. This material is not DNA.  Therefore, you can spin the insoluble material to the bottom of the tube and transfer the clear liquid to a new tube.
 

Setting up an analytical restriction digest

Setting up an analytical restriction digest.

Download and Excel file to automatically calculate  Master-mix volumes here. You could run your digests on slide agarose gels or standard agarose gels to resolve the DNA species. Slide agarose gels are faster and are my desired gel system for all DNA and RNA applications.

E. coli transformation and plating

Transforming E. coli with plasmids and plating the transformation.
 

I have found that the Inoue, H., Nojima, H., & Okayama, H. (1990). Gene, 96(1), 23-28. method gives the highest efficiency competent cells and is very easy to do. You can get a protocol for the Inoue method here.

Plant genomic DNA extraction

The CTAB method yields high quality plant genomic DNA that can be used for virtually any application including Illumina sequencing. The method is fast, cheap, and scalable. For genotyping, less than 1 mm x 1 mm pieces of plant material can be use.

Download a printable CTAB plant genomic DNA extraction protocol here.