Millipore - 96-well Plasmid and BAC Preparation Kit

Montage Plasmid Miniprep_HTS 96 Kit

The Montage Plasmid Miniprep_HTS 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 Miniprep_HTS 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.

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 Miniprep_HTS 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 Miniprep_HTS 96 Kit (MW-lambda Hind III digest).

High Yields*
Host strain	Plasmid	Yield (µg)
XL 1-blue	pUC19	2.1 – 6.1
XL 1-blue	pLH2**	3.5 – 9.2
JM109	pUC19	2.4 – 6.4
JM109	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 Hin*d 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
XL1-blue	pLH2	658	623
JM109	pUC19	706	602
JM109	pLH2	695	543
Sequence data from plasmids prepared with the Montage Plasmid Miniprep_HTS 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.

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 Miniprep_HTS 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 Miniprep_HTS 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 Miniprep_HTS 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 Miniprep_HTS 96 Kit is suitable for fingerprinting.

Compare	Catalogue No.	Description	Qty/Pk
	LSKP09604	Plasmid Miniprep₉₆ Kit	4	» Add to Cart
	LSKP09624	Plasmid Miniprep₉₆ Kit	24	» Add to Cart

Compare	Catalogue No.	Description	Qty/Pk
	LSKCCB050	Cell Culture Blocks with lids	50	» Add to Cart
	LSKP38450	384-well Plasmid Miniprep Clearing Plates	50	» Add to Cart
	SCGVU01RE	Stericup-GV, 0.22 µm, PVDF, 150 mL, radio-sterilized	12	» Add to Cart
	LSKAST100	Adhesive Tape Covers	100	» Add to Cart

Compare	Catalogue No.	Description	Qty/Pk
	MSVMHTS00	MultiScreen_HTS Vacuum Manifold	1	» Add to Cart
	LSKVBP100	V-bottom Plates	100	» Add to Cart
	WP6111560	Chemical Duty Pump, 115 V/60 Hz	1	» Add to Cart
	WP6122050	Chemical Duty Pump, 220 V/50 Hz	1	» Add to Cart
	WP6110060	Chemical Duty Pump, 100 V/50–60 Hz	1	» Add to Cart

Compare	Catalogue No.	Description	Volume, mL
	LSKCRS500	Cell Resuspension Solution 0.5 L	500	» Add to Cart
	LSKCLS500	Cell Lysis Solution 0.5 L	500	» Add to Cart
	LSKNS0500	Neutralization Solution, 0.5 L	500	» Add to Cart
	LSKNF0500	Nuclease-free Water, 0.5 L	500	» Add to Cart
	LSKCTB500	Tris Buffer, 0.5 L	500	» Add to Cart
	LSKPMRN30	RNase A, 0.9 mL	0.9	» Add to Cart