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

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 [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
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