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.
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 . Cosmid structures were created from a hybrid of both phage and plasmid traits . 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  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
Restriction digests were carried out to a final volume of 15 l using BamHI  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 .
Results and Discussion:
The ability of the Perfectprep Plasmid Mini to isolate large size cosmids
is shown by the restriction digest
s 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 . 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
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.
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.