Looking at Transfection Efficiencies

Introducing foreign DNA into mammalian cells is vital to many areas of research. The ability to study the effect of introducing a gene or oligonucleotide of interest into a cell line may depend on the effectiveness of the transfection reagent. Stratagene’s GeneJammer^® transfection reagent and LipoTAXI^® transfection reagent can achieve efficient transfections in a variety of cell lines. You can determine the optimal combination of transfection reagent and plasmid DNA by following simple protocols. However, mycoplasma contamination can result in low or atypical transfection efficiencies

In a typical protocol for optimizing transfection conditions, the amount of LipoTAXI reagent is increased at various concentrations of plasmid DNA that encodes for a reporter gene. Expression of the reporter gene increases until amounts of lipid or DNA prove toxic. While performing this optimization procedure in HeLa cells, we initially found unexpected results. The graph of our optimization procedure lacked the expected profile, and expression of the reporter molecule seemed unusually low and uncharacteristically flat (Figure 1).

Simple PCR Assay to Detect Mycoplasma Infection

We used the MycoSensor PCR assay kit to reveal infection in the HeLa cells (Figure 2). The kit features a reduced cycling time that allows you to detect mycoplasma contamination within three-and-a-half hours, including sample preparation and gel electrophoresis (Figure 3). An endogenous internal control reduces the chance of false-negatives by confirming that a successful PCR reaction has taken place.

Figure 3
The MycoSensor PCR Assay Kit Protocol

Maintain High-Efficiency Transfection

When optimizing transfection conditions using a transfection reagent, low or atypical results may indicate contamination. You can use the MycoSensor PCR assay kit to maintain high-efficiency transfection by testing for mycoplasma infection.

* Patent pending

See license reference 3

The MycoSensor PCR assay kit detects the eight most common species of mycoplasma and acholeplasma (Table 1). The kit includes a primer set, two positive controls, an internal amplification control, a dNTP/dUTP mix, and StrataClean™ resin. Just add your choice of DNA polymerase (we recommend Taq). The kit primers can detect infections from both supernatant and cell extract samples. The primers are designed to prevent the amplification of E.coli gDNA templates that sometimes contaminate recombinant Taq polymerase preparations. The two positive control templates validate that a polymerase-mediated amplification has occurred and confirm the size of the mycoplasma PCR product in the test samples. If the test cell line is infected, the mycoplasma primer mix yields a single 315-bp amplification product, regardless of which species of mycoplasma is present.