Protein expression and purification Mammalian cells HeLa

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Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Product

Get tips on using Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE to perform RNA isolation / purification Tissue - human kidney tissue

Products Fisher Scientific Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE

Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Product

Get tips on using Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE to perform DNA isolation / purification Bacteria - Gram positive Lactobacillus

Products Fisher Scientific Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE

cDNA synthesis Tissue Experiment

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.

RNA cDNA synthesis Tissue

cDNA synthesis Yeast Experiment

RNA cDNA synthesis Yeast

PCR Multiplex PCR - Bacterial DNA Experiment

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. Multiplexing such a reaction amplifies the design challenges where one target requires 3 primers, which should be exclusively bound nowhere in the template DNA or to each other. Similarly, two targets require 6, three require 9, and so on. Each amplicon needs to be either a different size (for gels) or labeled with a different fluorescent tag that is spectrally distinct from the others in the reaction. Further complicating this, different targets in the reaction can compete with each other for resources and causes more challenges in the detection of amplicons. However, with proper primer designing, their validation, optimize quality and concentration of the enzyme and buffers certainly lead to a successful multiplex PCR reaction.

DNA PCR Multiplex PCR Bacterial DNA

CytoSelect™ 24-Well Wound Healing Assay Product

Get tips on using CytoSelect™ 24-Well Wound Healing Assay to perform Wound healing assay cell type - rat MSC