Notes: To sequence the mitochondrial genome one of the most widespread and common collembolan species of Antarctica, springtail Cryptopygus antarcticus. Specimens were collected from Killingbeck I during a 2002 polar expedition and frozen in liquid nitrogen. The Wizard^® SV Genomic Purification System was used to extract total DNA from the samples and the complete mitochondrial genome was amplified twice, first with universal primers and sequenced, and then using long PCR with specific primers. The long PCR products were mechanically sheared, blunt end repaired and purified using the Wizard^® SV Gel and PCR Clean-Up System. The fragments were then cloned, transformed and sequenced. (3976)

Notes: A panel of 11 Gram-negative and 11 Gram-positive bacterial species was used to develop a real-time PCR detection method. Initially DNA was purified from 1ml of various dilutions of bacteria resuspended in a saline solution using either the QIAamp DNA blood mini kit or the Wizard^® SV Genomic DNA Purification System. The results suggested that the Wizard^® SV Genomic DNA Purification System extraction protocol was superior in disrupting the bacterial cell wall (especially of Gram-positive bacteria), to allow release of bacterial DNA. Using DNA purified with the Wizard^® SV Genomic DNA Purification System, detection of S. aureus and E. coli at concentrations as low as 10¹ CFU per PCR was achieved. The Wizard^® SV Genomic DNA Purification System purification protocol provided in eNotes online (www.promega.com/enotes/applications/ap0051_tabs.htm) was used with the following modifications: The bacterial pellet was resuspended in 400µl of enzymatic lysis solution (47mM EDTA, 25mg/ml lysozyme, 20µg/ml lysostaphin) and incubated for 2 hours at 37°C. Next, 19.2mg/ml proteinase K was added (final concentration 0.4mg/ml), and the mixture was incubated for 1 hour at 55°C. Finally, the Nuclei Lysis Solution and RNase Solution were added, mixed and incubated for 10 minutes at 80°C. For real-time PCR analysis, 2µl of genomic DNA was used. (3676)