The HotSHOT DNA Extraction Kit provides users with a rapid, inexpensive, and reliable alkaline lysis method to extract PCR-quality DNA from a wide range of DNA-rich tissues. Suggested target tissues include animal tissues (from mammals to fish to invertebrates), although this protocol has also often been used successfully to extract fungal, plant, and bacterial DNA in a number of studies. It is a relatively crude DNA extraction method and is therefore not suitable for tissue types containing high proportions of PCR inhibitors.
This protocol was originally developed by Truett et al. (2000) for routine genotyping of mouse tissues. Since then it has been widely used in many different applications because of its low cost, simplicity, and scalability.
Extraction of PCR-grade DNA from a wide range of sample types, for genotyping, PCR-based assays, sequencing, and DNA barcoding.
The HotSHOT alkaline lysis method most often used for extracting DNA from mammal, bird, and fish tissues, but have also been used on a wide range of tissues from different taxonomic groups, ranging from invertebrates, animal parasites, and parasite blood meals; zooplanktonic eggs; pollen, and young leaves; and fungal pathogens in plant tissues.
Add 75 µL of Alkaline Lysis Solution to 1-2 mm3 of a sample in a 0.2 mL PCR tube, and incubate at 95 ºC for 30 minutes. Allow to cool and then add 75 µL of Neutralising Buffer. Use 1–5 µl of DNA extract as a template per 20 µl PCR reaction.
Alkaline Lysis Solution (25 mM NaOH, pH 12)
Neutralising Buffer (100 mM Tris-HCl, 0.5 mM EDTA, pH 8)
Storage & Stability
Store sealed and in the original bag at room temperature or 4 ºC.
Storage for up to 6 months at room temperature should have minimal detrimental effects provided the containers and bags are properly sealed.
Exposure of the Alkaline Lysis Solution to air will gradually decrease the pH and effectiveness of this reagent over time. For longer term storage freeze at –20 ºC.
Shipped at room temperature.