GeneJET™ Plasmid Miniprep Kit From Fermentas

GeneJET™ Plasmid Miniprep Kit From Fermentas
In our lab, we routinely use the make-it-yourself Easyprep method [BioTechniques (1993) 14, 524-528] to screen large numbers of clones because it only requires one solution and is inexpensive. The Easyprep method results in DNA which cuts well with most restriction enzymes, but gives variable results with sequencing and other sensitive applications (e.g. RNAi, transfection). For these applications, we have tried a number of miniprep kits with mixed success (Qiagen's Miniprep Kit and Eppendorf's FastPlasmid Kit); we had problems obtaining sufficient yields and decent sequencing results. Thus, I decided to try Fermentas' GeneJET™ system because of the much lower price ($192 for 250 preps), its touted 14 minute prep time, and purity for downstream applications.

GeneJET™ is an alkaline lysis system. The solutions are labeled with clear terminology (Resuspension, Lysis, Neutralization, and Wash). You'll need to add the RNAse A solution to the Resuspension Buffer, and ethanol to the Wash Solution, before starting. Ethanol is not included; we obtain molecular biology grade absolute ethanol from Fisher Scientific. In my trial run, I prepared DNA from two 2ml cultures. The pellets were resuspended in Resuspension Buffer, lysed, and neutralized. After centrifugation, the supernatant was spun through the mini column. Following a wash step, the DNA was eluted with 50ul of the provided elution buffer into a new tube (not included in the kit). Processing 2 samples, from bacterial culture to DNA, took 24 minutes. On agarose gels, the DNA generated a single clear band and there was no RNA. The yield was 0.2ug/ul for a total of 10ug of plasmid per prep. This is less than the 20ug described in the instructions, but is still enough for most applications. 1ug of each DNA prep was used in automated sequencing and gave beautiful sequences up to 700bp. I also used the DNA together with Cellfectin (Invitrogen) to transfect Drosophila Kc tissue culture cells. The DNA worked well for transient expression and in the production of stable lines. The plasmids also functioned perfectly as templates for PCR reactions.

Users of the Qiagen Miniprep Kit may notice a strong similarity between the two kits. However, the Fermentas system is significantly cheaper (currently $192 vs. $302 for 250 preps), the solutions are labeled in plain English (as opposed to P1, P2, QC, EB, etc.) and the instructions are easier to read and include a troubleshooting section. Furthermore, the GeneJET™ mini spin columns come with a built-in cap (Qiagen's have no cap; Eppendorf has the cap on the collecting tube, but not the spin column.) GeneJET™’s cap on the mini spin column has the benefit of providing protection from contamination as well as providing a writing surface for labeling the sample. However, it does prove awkward during the final elution step when both the mini spin column and the collecting tube contain caps. This may also cause problems in the centrifuge with caps breaking off or with fitting the columns and tubes into the centrifuge.

I'll be using the GeneJET™ Plasmid Miniprep Kit in the future to prepare plasmids for sequencing and transfection. The method is too laborious, though, for large scale screening of clones, for which I recommend the Easyprep method (see above.)

Byron Williams
Research Associate
Cornell University
Department of Molecular Biology and Genetics

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GeneJET™ Plasmid Miniprep Kit From Fermentas
The Good

Clear instructions; clearly marked solutions; cap on mini spin column is a plus; quality of DNA is excellent for PCR, sequencing and transfection.

The Bad

Too many steps; need to provide ethanol; took longer than advertised.

The Bottom Line

If you have only a few preps to do, it's an excellent kit for plasmid minipreps of high quality.

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