( Isolation of Cosmids using Eppendorf's...)Isolation of Cosmids using Eppendorf's Perfectprep Plasmid ,,, Mini Kit

Isolation of Cosmids using Eppendorf's Perfectprep Plasmid Mini Kit

James Griffith and Kim Vollmer
James Griffith, University of Georgia, Department of Genetics, Athens, GA
Kimberly Vollmer, Brinkmann Instruments, Biosytems Application Lab, Westbury, NY Abstract

Cosmids are commonly used vectors in the lab, providing the capability to hold large fragments of DNA. They often help when creating genomic libraries where small plasmids and large constructs such as BAC's are not appropriate. Purification of cosmids is a necessary step during their use; Eppendorf's Perfectprep Plasmid Mini Kit is a rapid and effective way of purifying cosmid DNA from bacterial culture.

Introduction

Cosmids have become important tools for molecular biologists, filling a void left between small plasmid vectors and large vector constructs such as BAC's. Large vectors such as cosmids are extremely useful in creating genomic libraries with a capacity to hold fragments of DNA ranging from 30kb and 45kb DNA [1]. Cosmid structures were created from a hybrid of both phage and plasmid traits [2]. The intrinsic characteristics of cosmids allow them to undergo efficient ligation, transfection, replication and selection; providing the exclusivity needed for creating genomic libraries. The characterization of DNA inserts ligated into cosmids is one of the most common downstream applications when creating a library. Due to the size of cosmids it is important to find a method that will purify the cosmid without shearing or excessive loss of DNA. Eppendorf's Perfectprep Plasmid Mini Kit is very versatile, providing an easy and efficient way of isolating cosmids from bacterial culture without shearing or significant loss of product due to the binding properties of cosmids.

This article shows the capability of the Perfectprep Plasmid Mini Kit in isolating several cosmid constructions for screening purposes. A specially designed vector designated pLorist6Xh [3] was used in this experiment for screening of the Neurospora crassa genome. Restriction analysis of the pLorist6Xh vector isolated from the Perfectprep Plasmid Mini Kit was performed for analysis of DNA. The procedure shows the ability of the Perfectprep Mini Kit to isolate the pLorist6Xh vector containing inserts ranging from 1842kb.

Materials and Methods

• Bacterial Culture
  DH5a was used as the host strain for cosmid insert pLorist6Xh. Selected colonies were inoculated in 3 mls of LB with kanamaycin at a final concentration of 50 mg/ml. The cultures were placed in 14 ml snap cap tubes and allowed to grow at 300 rpm and 37C for 1416 hours, optimal growth will give OD 600 readings of an 1.52.0.
• Purification
  Cells were harvested at 4000 rpms for 6 minutes at room temperature in a 5810 centrifuge with rotor A-4-62. Removing supernant, harvested cells were resuspended in 100 ml of solution 1 (see Protocol below). Vortexing of sample allowed for complete resuspension. Aliquots of 1 ml samples were transferred to 1.5 ml tubes. The purification process was completed occurding to the Perfectprep Mini protocol below. Elution was carried out with 70 ml of elution buffer heated to approximately 60C to elute cosmid DNA.

Perfect Prep Mini Protocol:

1. Pellet 1 to 3 ml bacterial culture by centrifuging for 20 seconds. Remove supernatant by aspiration or decantation.
2. Add 100 l Solution I to the pellet and completely resuspend cells by vigorous vortexing.
3. Add 100 l Solution II and mix well by repeated gentle inversion.
4. Add 100 l Solution III and mix well by repeated vigorous inversion.
5. Centrifuge lysate for 30 seconds and transfer supernatant to a spincolumn.
6. Add 450 l well-mixed DNA Binding Matrix Suspension; mix well.
7. Centrifuge for 30 seconds. Decant filtrate from collection tube.
8. Add 400 l Diluted Purification Solution to the spin column; shake briefly. Centrifuge 60 seconds.
9. Decant filtrate and place spin column back in the collection tube. Spin for 60 seconds.
10. Transfer spin column to a fresh collection tube. Add 50 to 70 l of Elution Buffer to DNA Binding Matrix. Vortex briefly.

Centrifuge for 60 seconds, Discard used spin column and store eluted plasmid DNA at either +4C or 20C.

Analysis

Restriction digests were carried out to a final volume of 15 l using BamHI [4] and 7.5 l of each purified sample. The reactions were incubated at 37C for 1 hour. Electrophoretic analysis was carried out using all 15 ls of restriction digests loaded on a 0.7% Amresco Agarose III gel and run at 100 V for 2.5 hours. Two DNA standards were also loaded on the gel; l HindIII and 1 kb ladder [5].

Results and Discussion:

The ability of the Perfectprep Plasmid Mini to isolate large size cosmids is shown by the restriction digests of several different cosmid constructs within the [Neurospora crassa] library (Figure 1). Restriction digests, were used to determine quality, size and yield of plasmid DNA isolated from the Eppendorf's Perfectprep Plasmid Kit. The cosmid constructs examined in this library contain an insert size ranging from 1842 kb; further studies have shown the pLorist6Xh vector has an average insert size of 34 kb [3]. The results seen in Figure 1 show the capability of the Perfectprep Plasmid Mini Kit to purify high quality cosmid DNA. Inhibition of restriction endonucleases can occur if purified DNA is contaminated with impurities such as proteins or phenols. No inhibition or partial of digestion can be seen with the cosmids isolated with the Perfectprep Mini Kit. Additionally, DNA isolated with the Perfectprep Mini Kit can be used for other downstream applications with high success, such as transfection and sequencing (not shown).

Figure 1: Lane 1, restriction digest of H11F02 with BamHI; Lane 2, 1 Kb ladder; Lane 3 Hind III Marker; Lane 4 7, digestion of H115H12, H130A02, H104E08, H107G01 consecutively with BamHI; Lane 8, 1 Kb ladder; Lane 9 HindIII Marker; Lane 10, digestion of H9305 with BamHI The quantity of DNA was estimated using 500 ng of Hind III loaded in lane 3 and 9 of Figure 1. It is important to note when determining optimal yield for this experiment, that the pLorist6Xh vector is a low copy plasmid due to its origin of replication [3]. The amount of DNA sample loaded per well is about 1/10th the total amount, showing the ability of the Eppendorf Perfectprep Plasmid Mini Kit to provide good consistent yields per well ranging from 110 g.

Conclusion:

The restriction digest seen in Figure 1 represents the ability of the Eppendorf's Perfectprep Plasmid Mini purification system to purify large construct DNA vectors. The simplicity of the Perfectprep Mini protocol (see Materials and Methods) offers the ability to isolate quality cosmid DNA in high quantities, easily and efficiently. The DNA obtained can be used for several downstream applications other than the one represented here, including transfection and sequencing. Eppendorf's Perfectprep Mini Kit is a simple solution to the diversified needs of researchers involved with DNA vector isolation.

ACKNOWLEDGEMENTS:

I would like to say thank you to James Griffith for his work and results. To Joel Lopez and Vince Prezioso, I would like to say thank you for all your help downstream.

REFERENCES:

1. Ausubel, F.M., et.al., Current Protocols in Molecular Biology Vol. 1, Wiley and Sons, New York, 2001.
2. Hartwell, L.H., et.al., Genetic, From Genes to Genomes, McGraw Hill, New York, 2000.
3. Kelkar, H.S., et.al., The Neurospora Crassa Genome: Cosmid Libraries Sorted by Chromosome, Genetics 157, 2001, 979-990.
4. New England Bio Labs, Beverly, MA.
5. Promega Corporation, Madison, WI.

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