Molecular Biology

Rapid DNA Isolation and PCR Analysis Using FTA Paper

FTA paper is an attractive alternative to the standard methods for DNA isolation and storage. Using our fast protocol, a single person can isolate and analyze thousands of DNA samples in just few weeks. It worked great for genetic mapping with PCR markers and for genotypic analysis of segregating populations of T-DNA insertional mutants of Arabidopsis. Download the protocol: FTA_paper_processing.pdf

Filtering Plant Homogenates for Protein Gel Loading

Ground-up plant tissue samples tend to clog the protein gel loading tips. A simple and inexpensive home-made filter removes the debris, allows reproducible loading of desired volumes and makes protein gel loading almost a pleasure. Download: Micro-filter.pdf.

Rapid Screening for Plasmids with Cloned Inserts

When the traditional cloning does not work as expected and the background is high, it may be necessary to screen many colonies to find a clone with an insert. Clones with inserts can be detected by running uncut plasmids from lysed bacteria on agarose gels. About 200 bacterial colonies can be screened in one afternoon. Download: plasmid_screen.pdf

Blunt-end Ligation in the Presence of PEG

This protocol is based on information from Dave Shintani, University of Nevada, Reno.

2x ligation buffer (stored at -20 °C)

  • 100 mM Tris-HCl pH 7.5
  • 20 mM MgCl2
  • 10 mM DTT
  • 30% PEG 8000 (w/v)

10 mM ATP (store at -20°C) in 30mM tris-HCl buffer pH 7.5

Ligation reaction:

  • Vector DNA (15-60 fmol)
  • Insert DNA (50 - 200 fmol)
  • 10 µL 2 x ligase buffer
  • 1 µL 10 mM ATP
  • 1 µL T4 DNA Ligase (1U/µL)
  • H2O to final volume 20 µL

Incubate at room temperature several hours. For sticky end ligations, use less DNA and Ligase. Use for heat-shock transformation of chemically competent E. coli, use no more than 3 µL of the ligation reaction per 100 µL competent cells - if using more, PEG would decrease transformation efficiency.

Ligation in the Presence of a Restriction Enzyme

When cloning blunt-ended PCR products into non-dephosphorylated vectors (e.g. cut with SmaI), presence of the same enzyme in the ligation reaction helps reduce background and increase ligation efficiency by linearizing recircularized vectors. Of course, your blunt-end insert should not have the SmaI restriction site..

  1. To clone into a SmaI site (CCC|GGG), make sure your primer´s 5´ end is not GGG.
  2. Digest your vector with SmaI at 25°C for several hours or overnight, check for complete digestion on a gel (optional), and add your (non phosphorylated) PCR product.
  3. Perform a standard ligation reaction (10 µl, 1x ligase buffer, 1 unit ligase) but also add 5 units SmaI (0.5 µl). Incubate overnight on the bench.
  4. Dilute the 10 µl ligation reaction to 30 µl total volume (add 18 µl water and 2 µl appropriate restriction buffer) and then add another 10 units (1 µl) of SmaI for one hour.
  5. Use 5 µl of this reaction to transform E. coli competent cells.

For enzymes other than SmaI, check for the optimal conditions of the enzyme first. If it is expected to not work in ligase buffer, you can instead use the enzyme´s special buffer and add 0.5 to 1mM ATP. In some cases, it may not be possible to find conditions compatible with both enzymes (e.g. high temperature REs), so ligation must be performed first.