Protein Expression Eukaryotic cells N. benthamiana

Get tips on using Nucleic Acid Purification to perform Plasmid Isolation DH10Bac (Bacmid)

Get tips on using AvaII NEB#R0153 to perform Restriction Enzymes AvaII / Eco47I / VpaK11BI

Get tips on using AsiSI NEB#R0630 to perform Restriction Enzymes AsiSI / SfaAI / SgfI

Get tips on using AfeI NEB#R0652 to perform Restriction Enzymes AfeI / Eco47III / Aor51HI

Get tips on using HinP1I NEB#R0124 to perform Restriction Enzymes Hin6I / HinP1I / Hhal

Get tips on using AvrII NEB#R0174 to perform Restriction Enzymes BlnI / AvrII / XmaJI

The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.

DNA isolation and purification is the first critical step in sample preparation that helps ensure optimal performance of downstream assays like PCR, microarrays, and sequencing. Failure in yielding high-quality DNA would be the major reason that DNA doesn't work for the downstream application. To circumvent this, one should follow the recommended storage conditions to minimize DNA degradation by nucleases and shouldn't overload the purification system.