After removing the gel solution from the microwave we keep it for cooling for nearly 10 to 15 minutes so that the temperature reduces to 55-60 degrees and then only add EtBr. Why can't we directly add EtBr to the gel directly after removing it from the microwave or just 5 mins after removing from the microwave because it is stable upto 260 degrees and doesn't decompose upto that.
You can add it if you want to. We generally do not add Ethidium bromide into agarose if some agarose (and Ethidium bromide) was left in the bottle from previous preparation. This suggests that Ethidium bromide can be boiled together with agarose and it continues to bind with nucleic acid.
Thank you Sir. Now from your research experience one thing I clearly understood is etbr does not degrade when boiled with the gel but still I am trying to find out why people add it only when the gel cools down to 55-60 degree Celsius. Can it even slightly vaporize at 100 degrees and inhaled? Thank you again for sharing your research experience. I am really grateful to you
Dear Anirudha
The reason for allowing the agarose to cool a little before adding EtBr is to minimise production of ethidium bromide vapour which are toxic. Therefore adding it at 55-60C as you have been doing will be a safer option.
Cheers!
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