Site Directed Mutagenesis (SDM) Human Insertion IMR-32

Get tips on using Recombinant Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) to perform Immunohistochemistry Mouse - ERα

Get tips on using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit to perform DNA isolation / purification Cells - Immortalized cell lines SH-SY5Y

Get tips on using GenJet™ In Vitro DNA Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines MCF-7

Get tips on using X-tremeGENE™ HP DNA Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines PANC-1

Get tips on using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit to perform DNA isolation / purification Cells - Immortalized cell lines HEK 293T

Get tips on using GeneRead Size Selection Kit (50) to perform Whole Genome Amplification NGS library purification

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.