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Thermo Fisher Scientific Avoiding RNase Contamination User guide

  • Hello! I've reviewed the user guide for 'Avoiding RNase Contamination' from Thermo Scientific. This document provides essential protocols for maintaining an RNase-free environment during RNA work, focusing on aspects like reagent quality, sterile handling techniques, and solution preparation. I'm here to help answer your questions about these procedures and ensure your experiments are successful.
  • What is the main purpose of this document?
    What should be used to prepare buffers containing Tris?
    How should solutions containing DTT or nucleotides be prepared?
    What should be done with water and solutions used for RNA purification and handling?
Protocol
Avoiding RNase Contamination
Reagents have been tested to ensure they are endo-,
exodeoxyribonuclease, ribonuclease, and phosphatase
free. However, an RNase-free working environment
and RNase-free solutions are also critical factors for
performing successful in vitro transcription.
General recommendations to avoid RNase contamination:
• Maintain a separate area, dedicated pipettors and
reagents when working with RNA.
• Wear gloves when handling RNA and reagents to avoid
contact with skin, which is a source of RNases. Change
gloves frequently.
• Use sterile RNase free plastic tubes.
• Treat water and all solutions used for RNA purication
and handling with DEPC. Add DEPC to 0.1% (v/v)
nal concentration; incubate overnight at room
temperature and autoclave.
• High quality reagents must be used for buffer solutions.
Buffers containing Tris should be prepared by dissolving
Tris base in DEPC-treated water. Solutions containing
DTT or nucleotides should be prepared by dissolving in
DEPC-treated water and passing the solution through a
0.2 µm lter to sterilize.
• Keep all kit components sealed when not in use and all
tubes tightly closed during the transcription reaction.
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This protocol is for Avoiding RNase Contamination
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