CHO Lec3.2.8.1 cells have 4 independent mutations in the N and O glycosylation pathways. When cultured with alpha-glucosidase I inhibitor N-butyl-deoxynojirimycin, glycoproteins produced in CHO Lec3.2.8.1 cells are completely susceptible to Endo H digestion. Endo H cleaves chitobiose, leaving a single N-linked N-acetylglucosamine per site which is ideal for maintenance of protein solubility and special carb-protein interactions, such as between the first N-acetyl glucosamine residue and tryp. - [Read Establishment of Stable Transfectant of CHO Lec Cells Protocol]
In this protocol, double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]
Protocol describes how double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]
In the first part of this protocol, the linear range of amplification is determined by carrying out 10 identical PCRs in the presence of [{alpha}-32P]dCTP and stopping one reaction after every two cycles. Amplification products are quantified on a denaturing polyacrylamide gel and the results plotted on a graph (counts per minute vs. cycle number). Total RNA is used as an internal control. - [Read Relative RT-PCR: Determining the Linear Range of Amplification and Optimizing the Primers:Competimer]