DNA transfection Mammalian cells Immortalized cell lines

Get tips on using pX330-U6-Chimeric_BB-CBh-hSpCas9 to perform CRISPR Mouse - Deletion ES (embryonic stem) cells Slx2

Get tips on using pSpCas9(BB)-2A-GFP (PX458) to perform CRISPR Mouse - Deletion ES (embryonic stem) cells MIR

Get tips on using pIEX-4-Crypα-6His-mMBP-UMODpXR to perform Protein Expression Eukaryotic cells - S. frugiperda mMBP

Get tips on using TruSeq RNA Library Prep Kit v2 to perform RNA sequencing Rat - Retinal ganglion cells (RGCs)

Get tips on using TruSeq RNA Library Prep Kit v2 to perform RNA sequencing Mouse - ESCs (Embryonic Stem Cells)

Get tips on using Quant-iT™ RNA Assay Kit to perform RNA quantification Fuorimetric - human metastatic melanoma cells

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.

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.

Get tips on using DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit to perform ROS assay cell type - mouse splenocytes

Get tips on using DCFDA / H2DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit to perform ROS assay cell type - C2C12