Veriti 96 well thermal cycler long pcr system

Total DNA was isolated from 3T3-L1 adipocytes using DNA extraction kit. Based on the idea that DNA damage blocks DNA polymerase progression [66 (link)]. PCR was employed in detection of DNA damage. The long fragment target DNA was amplified with Long Amp Taq Master Mix polymerase, while the short fragment target DNA was amplified with TaKaRa Taq DNA polymerase (primer sets listed in Table 2). The PCR reactions were performed in Veriti 96 well thermal cycler long PCR system (Applied Biosystems, Foster, CA, USA) as previously described [51 (link)]. After amplification, the PCR products were resolved in 1.0% and 0.5% agarose gel and visualized by staining with ethidium bromide. The long/short mtDNA ratio was calculated based on intensity of bands.

Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total genomic RNA, and the quality of isolated RNA was determined by spectrophotometry (260 nm). Reverse transcription was performed with total RNA (1 μg) and the M-MLV Reverse Transcriptase Kit (Promega A3500; Promega, Madison, WI, USA). Briefly, the total reaction volume (40 μL) was used in a Veriti 96 Well Thermal Cycler long PCR system (Applied Biosystems, Foster City, CA, USA) with the following reaction conditions: 72 °C for 3 min, 42 °C for 90 min, 70 °C for 15 min, and hold at 4 °C.

Total genome DNA or RNA was isolated with a DNA extraction kit (TIANGEN, Beijing, China) or Trizol reagent (Invitrogen, Carlsbad, CA, USA), respectively. The quality of isolated DNA or RNA was determined by spectrophotometry at 260 nm. Reverse transcription was performed with 1 µg total RNA and M-MLV Reverse Transcriptase Kit (Promega A3500; Promega, Madison, WI, USA). Briefly, 40 μL of total reaction volume was used in a Veriti 96 Well Thermal Cycler long PCR system (Applied Biosystems, Foster City, CA, USA) with the following reaction procedure: 3 min at 72 °C, 90 min at 42 °C, 15 min at 70 °C, and hold at 4 °C.