I am attempting to recreate a protocol from a colleague's notes but I'm missing some key info. Unfortunately the colleague has since left so I can't ask them directly.
We are using rabbit reticulocyte lysates for cell-free protein production. After a small scale 25uL production the notes say to add 3uL 10N NaOH and 5uL H2O2 and centrifuge for 10-20 minutes. Presumably this is to remove any RNases and precipitate them out.
However.... I do not have the stock concentration of the H2O2. I am not familiar with this protocol and Googling hasn't been helpful. Can anyone advise on the concentration of the H2O2 I am to add?
Hello,
I didn't see all the protocol you run, but this step is rather looking suitable for halting translation and probably degradadion of all the mRNA left in the reaction.
In this protocol (Article In Vitro Translation Using Rabbit Reticulocyte Lysate
) the final conc. of NaOH and H2O2 is roughly 1M and 2%.So in your case to reach final 2% in 33 ul with 5 ul of H202, you may start with approx. 13% stock.
Hope this will help.
UPD: I think that the whole idea of removing RNAse after the translation looks somewhat odd.
H2O2 usually comes as a stock solution, so in the absence of specific concentration information it most likely is just "straight from the bottle".
However RNA is degraded at alkaline pH and the amount of NaOH you are adding will make the pH highly alkaline so probably all the RNA will be degraded. I would guess that this is how the reaction is terminated after doing the protein synthesis and has nothing to do with removing RNAse.
Anatoliy Zubritskiy and Michael J. Benedik - thanks for your help! based on what you say I think I must have misheard and the idea is to remove RNAs not RNase! I had assumed it was protein as analyzing the samples by SDS PAGE had been giving a messy background, but good to know. Combining your advice I will try 5uL of a 13% H2O2 solution made from the standardized stock H2O2
Elizabeth Radley sometimes SDS PAGE messy background (typically around 60-70 kDa) may come from keratine in bMe, DTT or sometimes SDS.
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