Genomic dna isolation kit

Total genomic DNA was extracted from each group of adults (10 females and 10 males, respectively) from field or laboratory colonies, and from dissected tissues and organs using a Genomic DNA isolation kit (Qiagen, Hilden, Germany). The concentration of the extracted genomic DNA was measured using a Nanodrop (Thermo Fisher Scientific, Waltham, USA) and the purity was evaluated by electrophoresis of the extracts in 1% (w/v) agarose gels.

Cell survival was determined by detection of Sry gene using qPCR in the myocardial tissue samples. Tissue samples were snap-frozen in liquid nitrogen and powdered. DNA purification was performed using the Genomic DNA Isolation kit (Qiagen, Germantown, MD, USA), and the concentration of the purified DNA was determined by spectrophotometry. The primer sequences for sry gene and β-actin were as follows: sry gene, forward 5′-GAGGCACAAGTTGGCTCAACA-3′ and reverse 5′-CTCCTGCAAAAAGGGCCTTT-3′; β-actin, forward 5′-CCACCATGTACCCAGGCATT-3′ and reverse 5′-ACTCCTGCTTGCTGATCCAC-3′.

F. acidiphilum Y^T (DSM 12658^T) was deposited to the DSMZ collection in 1998, and since then maintained in the laboratory, in 2008 the original isolate was retrieved from DSMZ for genome sequencing. F. acidiphilum Y^T was routinely grown on the Medium 9 K containing 25 g/l of FeSO₄.7H₂O, supplemented with 0.02% (w/vol) of yeast extract until the mid-exponential phase at as described previously^{1 (link)}. For calculation of single substitution mutation rates, the 100-ml cultures were grown in Erlenmeyer flasks under optimal conditions^{1 (link)} since deposition of the strain to the DSMZ Strain Culture collection in 1998. As an inoculum, 10 ml of culture were used each time, with 164 repeated growth experiments. The final culture (2008) was subjected to the DNA extraction and sequencing. Isolation of DNA from both variants was conducted using Genomic DNA isolation kit (QIAGEN, Hilden, Germany).

Genomic DNA from the transformant cultures was isolated using a genomic DNA isolation kit as per manufacturer instructions (Qiagen, Valencia, CA). About 100 ng of genomic DNA recovered from cultured organisms was digested with BglII restriction enzyme for 2 to 3 h at 37°C. The digested DNA samples were resolved on a 0.9% agarose gel for about 6 h at 60 V and transferred to a nylon membrane. Blots were then hybridized with a ³²P-labeled aadA gene probe at 68°C overnight, followed by washing steps to identify specific DNA-probe interactions as per our previously described protocol (14 (link)). The hybridized membranes were exposed to X-ray film to observe radioactive signals emitting from hybridized blots.

DNA was isolated from whole blood containing Acid Citrate Dextrose (ACD) solution, using a Genomic DNA Isolation kit, according to the manufacturer's instructions (Qiagen). In brief, 20 μL of Qiagen protease and 200 μL of whole blood were added into a 1.5 mL microcentrifuge tube. 200 μL of buffer (AL) was added and then incubated at 56°C for 10 minutes, and 200 μL of ethanol (100%) was added and centrifuged. The mixture was transferred into a QIAamp mini spin column, the tube was centrifuged, the QIAamp mini spin column was placed in a clean 2 mL collection tube, and the tube containing the filtrate was discarded. 500 μL of buffer (AW1) was added and centrifuged, then the QIAamp mini spin column was placed in a clean 2 mL collection tube, and the tube containing the filtrate was discarded. The procedure was repeated with 500 μL of buffer (AW2), and then, the QIAamp mini spin column was placeed in a clean 1.5 mL microcentrifuge tube. Then, 200 μL of elusion buffer (AE) was added and centrifuged, the QIAamp mini spin column was discarded, and the microcentrifuge tube containing the eluted DNA was stored at −80°C.