Purelink genomic dna extraction

Genomic DNA was extracted with the kit Purelink Genomic DNA extraction (Invitrogen) according the manufacturer’s instructions and eluted in 10 mM tris (pH 7.8). A total of 30 ng of DNA was incubated in master mix containing bovine serum albumin (0.2 mg/ml); 0.1% Tween 20; 1 mM each of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate; 4 μM dithiothreitol; φ29 buffer 1×; and φ29 DNA polymerase in a volume final of 20 μl and incubated at 30°C for 8 hours and then at 65°C for 20 min. Each assay was performed with and without the φ29 DNA polymerase. A slot blot analysis in nondenatured condition was done to quantify each CC assay. The 20 μl of CC assay was diluted in 300 μl of 2× SSC and slot-blotted on positively charged membrane Hybond-XL (Amersham). After UV cross-linking (0.120 J), the membrane was hybridized for a minimum of 6 hours at 37°C in Perfect Hybridization Plus Buffer (Sigma-Aldrich) with labeled ³²P-(CCCTAA)₆ telomeric probe. The membrane was exposed for various times (2 hours to overnight) to the Phosphor screen and read by Phosphor Imager. Statistical analysis was performed using the Prism software with a Student’s t test. Asterisks denote P values below to 0.05, and “NS” denotes nonsignificant statistical test.