Sample Preparation |
Purification of Cloned DNA
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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).
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| Figure 2. Restriction analysis of fosmid DNA prepared using
the Direct Lysis Fosmid96 Kit. Randomly chosen clones from a fosmid library
were digested with Not I and the DNA was analyzed by agarose gel electrophoresis.
Lane M, Kilobase ladder; lane 1, 40-kb control insert; lanes 2-19, Not I-digested
fosmid DNA. |
Figure 3. Sequence analysis confirms fosmid DNA quality. Sequencing
was performed using 4 µl of DNA isolated from an autoinduced pCC1™
fosmid using pCC1Fos Reverse Sequencing primer, and was analyzed on an
ABI® 3730 Analyzer in the UWBC DNA Sequencing and Synthesis Facility (Madison,
WI). |
BACMAX96™ DNA Purification Kit
CopyControl™ BAC Cloning Kits (BamH I, EcoR I, or Hind III)
CopyControl™ pCC1BAC™ Cloning-Ready Vectors (BamH I, EcoR I, or Hind III)
CopyControl™ Fosmid Autoinduction Solution
CopyControl™ BAC Autoinduction Solution
CopyControl™ Fosmid Library Production Kit
CopyControl™ HTP Fosmid Library Production Kit
Direct Lysis Plasmid96 DNA Purification Kit
Fast-Link™ DNA Ligation and Screening Kit
FosmidMAX™ DNA Purification Kit
Plasmid-Safe™ ATP-Dependent DNase
TAQXpedite™ PCR System (FAST End-Point)
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