Sample Preparation |
Purification of Cloned DNA
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ASM poster
2008 (2.2M PDF)
Applications
- Rapid, direct-lysis preparation of plasmid DNA for sequencing in a 96-well
format.
The Direct Lysis Plasmid96 Kit isolates plasmid 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 either high-copy or single-copy
plasmids sized up to 40 kb (Figs. 2 and 3). The kit uses a modified alkaline
lysis procedure that incorporates a proprietary RNase Blend to remove the RNA
efficiently. The typical yield from a 200-µl overnight culture of a high-copy
plasmid vector is sufficient for up to 50 end-sequencing reactions. A copy-number-induced
40-kb plasmid will yield DNA for six to eight 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 150
ml with a standard 1.2-ml prep.
- Low final DNA suspension volume (50 µl).
- Protocol complete in as little as 25 minutes.
- Perform ~50 end-sequencing reactions from a high-copy plasmid, 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. Examination of the quality of plasmid DNA. DNA
was isolated from 200-µl overnight cultures of randomly picked clones
from a shotgun library (2-6 kb) in a 96-well plate using the Direct Lysis
Plasmid96 Kit. A 5-µl aliquot of each 50-µl prep was analyzed
on a 1% agarose gel in 1X TAE buffer for 2 hours. Lane M, Supercoiled DNA
Ladder; lanes 1-18, supercoiled plasmid DNA. |
Figure 3. Sequence analysis confirms plasmid DNA quality. Sequencing
was performed using 2 µl of DNA isolated from a plasmid and M13 Forward
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 Library Production Kit
CopyControl™ HTP Fosmid Library Production Kit
Direct Lysis Fosmid96 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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