|DNA Template Prep and Sequencing Reaction Cleanup|
|Montage Plasmid Miniprep96 Kit|
|Can I use this kit in centrifugal mode?||Use of the Montage Plasmid Miniprep-96 Kit in a centrifugal protocol is not recommended.|
|I'm using your two plate plasmid prep protocol and the wells are overflowing when I transfer the cleared lysate to the FB plate. What am I doing wrong?||Overflowing can result from any of the following:
|I have observed RNA contamination of my plasmid preps. Why?||Failure to add RNase A to solution 1 will often result in RNA contamination in your DNA miniprep.|
|I have observed lower than expected yields for my plasmid minpreps when using the Montage kit. Why?||There are a number of factors that can contribute to low yields with the Montage kits they include: low/high copy number plasmid; bacterial strain: culturing time: inadequate cell resuspension: and overdrying of plasmid plate.|
|I have observed that the wells of the MultiScreen plate used in the Montage kit are not filtering uniformly. Why?||The wells of the MultiScreen plates usually filter uniformly. However, a bubble in the lysate may cause an airlock, which halts filtration. Also, a highly viscous sample may increase filtration time considerably.|
|What is holdup volume of a MultiScreen Plate used with the Montage Kits?||The holdup volume for the MultiScreen plate is between 5-20 microliters.|
|When using the Montage kits how can I tell when filtration in the MultiScreen plate is complete?||Filtration is complete when the individual wells of the MultiScreen plate are empty and the membranes appear shiny.|
|The two sections of the MultiScreen plates used in the Montage kits are separating under vacuum. Why?||The most common reason for this problem is the lack of a MultiScreen underdrain support grid in the vacuum manifold. A MultiScreen support grid is required for proper functioning of the MultiScreen plate with any vacuum manifold.|
|The total yields with the Montage Plasmid Miniprep-96 kit seems to be less then with competitive products. Why?||The Montage kit uses less culture volume (1ml vs. 1.3 ml) than most competitive kits. However the relative yield is equivalent.|
|What is the total processing time for plasmid preparation using the Montage kits?||Processing time depends on viscosity of sample, number of manifolds, familiarity with protocol, temperature. (Also, need vacuum capable of 24 in Hg or 812.7 millibar, 609.6 torr.)|
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|Life Science Research > Genomic Analysis > DNA Preparation & Cloning > DNA Purification > Plasmid Purification Kits & Reagents|
|Life Science Research > Cell Culture and Systems > Cell Culture Flasks, Plates, & Slides > Multiwell Plates|
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
|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 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 MiniprepHTS 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
Agarose gel showing quality and consistency of plasmid DNA purified using the Montage Plasmid MiniprepHTS 96 Kit (MW-lambda Hind III digest).
|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.
|Host strain||Plasmid||Average Read
q >20 bases
(n = 24)
|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.
Performance— BAC 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.