Dear colleagues,
I am facing a technical problem after chromatin fixation and shearing. In particular, it seems that the excess of formaldehyde used for fixation is inhibiting the RNAse, thus, my chromatin sample, which appears properly sheared on gel, presents a strong RNA band.
Have you ever experienced something similar? Do you have any suggestion to get rid of this formaldehyde excess?
Thank you in advance
Jacopo Biotti
Add Glycine: Glycine is commonly used to quench formaldehyde by reacting with it to stop crosslinking activity.
Hello Jacopo Biotti,
Bareera Zahoor is right. Glycine can quench formaldehyde because the amino group of glycine can react with formaldehyde, which prevents it from forming cross-links with other macromolecules. You may quench formaldehyde with 125 mM glycine for 5 mins at room temperature.
Even though glycine has been used routinely to quench crosslinking, Tris is a more efficient quencher, which can be explained chemically by the ability of Tris to form a cyclic product upon reaction with formaldehyde. However, at higher concentrations of Tris, which would likely be used for quenching, Tris can also facilitate crosslink reversal. If you do not succeed with glycine, use Tris.
For efficient quenching, use Tris pH 8.0 in a ∼2.25-fold molar excess. For 2% formaldehyde, you may use a final concentration of 1.5M Tris, and for 1% formaldehyde, use a final concentration of 750 mM Tris.
For more information, you may want to refer to the article attached below.
https://pmc.ncbi.nlm.nih.gov/articles/PMC4646298/
Best.
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