Journal: Nucleic Acids Research

Article Title: Transformation of isolated mammalian mitochondria by bacterial conjugation

doi: 10.1093/nar/gni140

Figure Lengend Snippet: T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.

Article Snippet: RT–PCR analysis Total RNAs from the T7RNAP-mitochondria mating mixture or from electroporated mitochondria were collected by isopropyl alcohol precipitation, and residual DNA contaminants were removed using RNase-free DNase I (Promega, Madison, WI) as directed by the manufacturer.

Techniques: Conjugation Assay, Blocking Assay, Reverse Transcription Polymerase Chain Reaction