Users' questions

Why is guanidine isothiocyanate used in the isolation of RNA?

Why is guanidine isothiocyanate used in the isolation of RNA?

Guanidinium thiocyanate is also used to lyse cells and virus particles in RNA and DNA extractions, where its function, in addition to its lysing action, is to prevent activity of RNase enzymes and DNase enzymes by denaturing them. These enzymes would otherwise damage the extract.

Does guanidine thiocyanate inhibit PCR?

If any GITC bleeds through into your DNA (or indeed RNA) ,which a 260/230 ratio of < 1.0 implies this chaotropic substance can definitely inhibit procedures (enzymes) like PCR .

How does guanidine thiocyanate denature proteins?

The guanidinium thiocyanate–phenol solution, which is commercially available as TRIzol, TriFast, or TRI Reagent, disrupts the cells, denatures the proteins, and deactivates the nucleases, thereby stabilizing the DNA, RNA, and protein. Another simple method is to use hot acid phenol to obtain RNA from microorganisms.

What does guanidine isothiocyanate do?

UltraPure™ Guanidine Isothiocyanate is a strong protein denaturant when used at high concentrations. It is useful in the isolation of intact RNA from sources rich in RNase such as pancreas (1).

How is guanidinium thiocyanate used in RNA purification?

The guanidinium thiocyanate-cesium gradient method of RNA purification described by Chirgwin et al. (1979) has been used successfully, as well as the acid guanidinium thiocyanate-phenol-chloroform extraction method ( Chomczynski and Sacchi, 1987 ), but other methods may also work.

When to use guanidine thiocyanate with phenol?

When used with acid-equilibrated phenol or phenol:chloroform, a solution of Guanidine Thiocyanate and beta-mercaptoethanol are very effective in disrupting both cytoplasmic and nuclear membranes while maintaining the integrity of the RNA. In addition, Guanidine Thiocyanate is also used as a storage buffer for blood samples.

How does guanidin thiocyanate work for humic substance removal?

The solution was clear, after extraction, so it seems to work for humic substance removal. However, does Guanidin Thiocyanate itself provide the problem that PCR reaction might be inhibited. Nanodrop resulted in low ratios for A260/A230. Can anyone give me some advice?

How to centrifuge Guanidinium thiocyanate to avoid interphase?

After cooling on ice for 15 min, centrifuge the sample at 10,000 g in a microfuge at 4°C. The aqueous phase (0.5 ml) containing the RNA is transferred to a second tube, avoiding any interphase. After the addition of 0.5 ml of 2-propanol, RNA is allowed to precipitate at −20°C for at least 1 hr.