Overnight culture: Add lysis buffer; mix. Add neutralization buffer; mix. No cell harvesting. No use of deep-well culture plate. Clear the lysate: Vacuum filtration or centrifugation, 1 min.   Precipitate the DNA: Centrifuge, 10 min. Wash with 70% EtOH, 5 min. Resuspend the DNA. Figure 1. Overview of the Direct Lysis Fosmid96 purification process.

ASM poster 2008, 2.2M PDF

Applications

• Rapid, direct-lysis preparation of fosmid DNA for sequencing in a 96-well format.

The Direct Lysis Fosmid96 Kit isolates fosmid DNA by direct lysis of a 200-µl overnight culture, eliminating the need for centrifugation of the culture and resuspension of the cell pellet (Fig. 1). The kit is designed for rapid preparation of sequencing templates in a 96-well format from fosmid clones (Figs. 2 and 3). The kit uses a modified alkaline lysis procedure that incorporates a proprietary RNase Blend to remove the RNA efficiently, and an autoinduction solution for higher cell growth and DNA yield when used with CopyControl™ fosmid clones. The typical DNA yield from a 200-µl overnight autoinduced culture of a CopyControl fosmid is sufficient for six to eight end-sequencing reactions.

The kit includes reagents and plastics for high-throughput DNA extraction, and is suitable for robotic automation procedures.

Benefits

• No cell harvesting or resuspension of the cell pellet.
• Inoculation of culture in a 200-µl volume eliminates the need for 2-ml, deep-well growth plates.
• Reduces the amount of media used to under 20 ml per plate compared to 115 ml with a standard 1.2-ml prep.
• Low final DNA suspension volume (25 µl).
• Protocol complete in as little as 25 minutes.
• Perform six to eight end-sequencing reactions from an autoinduced CopyControl fosmid (40-kb) clone, with average read length of 700-900 bases (Fig. 3).
• Low-profile filter-receiver plate block (H57 x L127 x W85 mm).