96-well Plasmid and BAC Preparation Kit

96-well Plasmid and BAC Preparation Kit Montage Plasmid MiniprepHTS 96 Kit The Montage Plasmid MiniprepHTS 96 Kit is a fast, easy-to-use kit for high-purity plasmid or bacterial artificial chromosome (BAC) minipreps. Using a patented separation technology, the Montage Kit follows a simple protocol that eliminates lengthy bind/elute methods and centrifugation steps to yield clean and reproducible DNA in 50% less time than traditional methods. Purified PCR samples are suitable for the most sensitive downstream applications including cloning, DNA sequencing, and transformation. BAC applications include sequencing, fingerprinting, arraying, and PCR amplification. The kit includes all the reagents and disposable materials you need to purify plasmid or BAC DNA in a 96-well format. Following bacterial lysis, three short filtration steps are all that are required to prepare 96 clean DNA samples from each plate. The Montage Plasmid MiniprepHTS 96 Kit is ideal for use with automated liquid handlers. Collection of purified sample from the top of the plate and the elimination of lengthy centrifugation steps enhance the kit's compatibility with a wide range of automated systems. - No centrifugation or precipitation steps required - Minimum processing time - Excellent purity, yields and reproducibility - Automation compatible

Kit Components

Part Function 96-well deep well culture block Growth of host bacteria Plasmid plate Purification of plasmid DNA Lysate clearing plate Clearing of bacterial lysate V-bottom storage plate Storage of plasmid DNA samples Sealing tape Sealing of V-bottom storage plate RNase A Required additive for Solution 1 Solution 1 Cell resuspension Solution 2 Cell lysis Solution 3 Neutralization Solution 4 Wash Solution 5 Resuspension/storage of plasmid DNA Foil seal Sealing plates during growth (must be pierced) For more information on kit components, refer to the Montage Plasmid MiniprepHTS 96 Kit operation manual or visit www.millipore.com/montage.

Protocol for Plasmid DNA Miniprep
For BAC DNA protocol, see Montage Plasmid Miniprep_HTS Kit user guide, P36315

1. Following bacterial lysis, transfer lysates from deep well culture block into clearing plate. Clear lysates into Plasmid filter plate using vacuum. 2. Move Plasmid plate to top of manifold and apply vacuum for 5 to 7 minutes to concentrate sample. Plasmid DNA is retained on the membrane surface while contaminants are filtered to waste. Add wash solution and filter for 3 to 5 minutes. 3. Add resuspension buffer and collect purified sample by aspiration. 4. Transfer samples to V-bottom plate for use or storage.

Performance — Plasmid DNA

High Quality Agarose gel showing quality and consistency of plasmid DNA purified using the Montage Plasmid MiniprepHTS 96 Kit (MW-lambda Hind III digest).

High Yields* Host strain Plasmid Yield (µg) XL 1-blue pUC19 2.1 – 6.1 pLH2** 3.5 – 9.2 JM109 pUC19 2.4 – 6.4 pLH2** 3.5 – 9.1 *Results will vary depending on host strain, plasmid, and protocol conditions. **pLH2 was derived from pUC19. The 2.0 Kb fragment of Hind III digested l phage was cloned into the Hind III site of pUC19, resulting in a 4.7 Kb plasmid.

Reliable Results Host Strain Plasmid Average Read Length (n=24; >98% accuracy) Average Phred q >20 bases (n = 24) XL1-blue pUC19 707 618 pLH2 658 623 JM109 pUC19 706 602 pLH2 695 543 Sequence data from plasmids prepared with the Montage Plasmid MiniprepHTS 96 Kit. All samples were sequenced using BigDye sequencing chemistry and were run on a MegaBACE® capillary array sequencer. 160 ng of pLH2 and 100 ng of pUC19 were used in the respective reactions. Reactions were cleaned up using Montage SEQ384 Kit.

Long Reads DNA template was prepared using the Montage Plasmid MiniprepHTS 96 Kit and used in BigDye sequencing reactions (1/16 scale). The reactions were cleaned up using Montage SEQ384 Kit and run on a MegaBACE 1000 sequencer.

Performance— BAC DNA

High Speed and High Performance Prep Time 56 minutes Average Yield (µg/well) 0.86 (± 0.28) Average Phred 20 bases 411 (± 152) Pass Rate (% wells with >100 Phred 20 bases) 90 Data were generated with 96 BAC clones (human chromosome 22 library, plate #4, Research Genetics, Inc.). BAC DNA was isolated from the 96 clones with the Montage Plasmid MiniprepHTS 96 Kit. Prep time starts with pelleted bacterial cells prior to lysis, and includes DNA resuspension time. DNA yield was measured in a fluorometric assay using SYBR® green I nucleic acid stain (Molecular Probes, Inc.). The DNA preps were sequenced using an optimized BigDye® version 3.0 (Applied Biosystems, Inc.) sequencing reaction setup and cycling conditions. Sequencing reactions were cleaned on Montage SEQ384 plates prior to injection on the ABI 3700 DNA sequencer. Pass rate is defined as the percentage of the 96 clones that yielded at least 100 Phred 20 bases.

BAC DNA purified with the Montage Plasmid MiniprepHTS 96 Kit was dissolved in 35 µL of the kit's Resuspension Buffer (solution 5) and 10 µL of the DNA were used for sequencing in a BigDye version 3.0 sequencing reaction (3/8 scale) as described in the kit's optimized protocol. The sequencing reaction was cleaned up using Montage SEQ384 plate and injected onto the ABI 3700 capillary sequencer.

Chromosomal DNA DNA from three different BAC clones from a human chromosome 22 human BAC DNA library (Research Genetics, Inc.) was purified with the Montage Plasmid MiniprepHTS 96 Kit and electrophoresed on a 0.8% agarose gel for 120 min at 100 V either uncut (lanes 1 – 3) or after digestion with Not I (lanes 4 – 6) or Hind III (lanes 7 – 9). Lane M is a Hind III digest of Lambda phage DNA. The BAC DNA purified with the Montage Plasmid MiniprepHTS 96 Kit is suitable for fingerprinting.