12 Quick Tips for NGS Library Preparation

Summary

This video offers twelve essential tips for next-generation sequencing (NGS) library preparation using any BEX products. It emphasizes the importance of spinning down vials, using filter tips, and maintaining aseptic techniques to prevent contamination. The script advises on cleaning work surfaces with RNA Zap, changing gloves frequently, and keeping samples and reagents capped. It also covers the proper handling of RNA, including storage conditions and avoiding freeze-thaw cycles. Additionally, it stresses the use of nuclease-free water, gentle pipetting to mix components, and the importance of keeping enzymes on ice for stability.

Takeaways

• 🌀 Always spin down vials before opening to prevent sample loss.
• 🛡️ Use filter tips to minimize contamination risks.
• 🧼 Practice aseptic techniques when handling RNA to avoid bacterial and mold contamination.
• 🧹 Clean your workspace with RNA zap or a similar product to reduce RNase contamination.
• 🧤 Change gloves frequently to further minimize contamination.
• 🔒 Keep sample and reagent vials capped when not in use to maintain sterility.
• ❄️ Keep RNA on ice and avoid freeze-thaw cycles to preserve integrity.
• 💧 Use nuclease-free water for dilution to prevent enzymatic degradation.
• 🌡️ Gently pipette up and down to mix reaction components without introducing air bubbles or shearing DNA/RNA.
• ❄️ Keep all master mixes on ice to ensure optimal enzyme activity before use.

Q & A

Why is it important to spin down the vials before opening them in NGS library preparation?

Spinning down the vials before opening ensures that all contents are at the bottom of the vial, preventing any from being in the cap or on the sides, which could lead to sample loss or contamination.

What is the purpose of using filter tips during NGS library preparation?

Filter tips are used to minimize contamination by preventing particles or microorganisms from entering the sample or reagents, which is crucial for the accuracy and integrity of the NGS results.

Why should aseptic techniques be used when working with RNA?

Aseptic techniques are essential when working with RNA to prevent contamination from bacteria and mold, which are common sources of RNase contamination that can degrade RNA and compromise the quality of the NGS library.

How can you minimize RNase contamination during NGS library preparation?

To minimize RNase contamination, one should clean working surfaces with RNA Zap or a similar product, change gloves frequently, and handle RNA samples with care to avoid introducing RNases from hands or dust particles.

Why is it necessary to keep sample and reagent vials capped when not in use?

Keeping vials capped when not in use prevents evaporation and contamination, ensuring the stability and integrity of the samples and reagents throughout the NGS library preparation process.

What is the significance of using RNase and DNase-free plastics and water?

Using RNase and DNase-free plastics and water is crucial to prevent enzymatic degradation of nucleic acids, which could otherwise compromise the quality and yield of the NGS library.

Why should RNA samples be kept on ice and avoid freeze-thaw cycles?

RNA samples should be kept on ice to maintain their stability and avoid freeze-thaw cycles, which can lead to RNA degradation and loss of sample integrity, affecting the outcome of NGS analysis.

What is the recommended storage temperature for purified RNA?

Purified RNA can be stored at either minus 20 degrees Celsius or minus 70 degrees Celsius to preserve its integrity and prevent degradation over time.

Why is it important to use nuclease-free water for dilution in NGS library preparation?

Using nuclease-free water ensures that there are no contaminating nucleases present that could degrade the nucleic acids during the dilution process, which is critical for maintaining the quality of the NGS library.

How should you mix reaction components to ensure efficient mixing without introducing shearing forces?

To efficiently mix reaction components without introducing shearing forces, one should gently pipette up and down rather than vortexing, as vortexing can cause shearing of the nucleic acids.

What is the reason for keeping all master mixes on ice and using them immediately after mixing?

Keeping master mixes on ice and using them immediately after mixing helps to maintain the optimal activity of enzymes and other components, which is essential for the efficiency and success of the NGS library preparation reactions.

Why should enzymes be returned to the freezer after use in NGS library preparation?

Returning enzymes to the freezer after use helps to ensure their stability and maintain their activity for future use, as enzymes can lose their effectiveness if not stored properly.

How should the final library be stored to ensure its stability?

The final library should be stored in low bind DNA tubes with TE buffer to minimize the risk of DNA or RNA binding to the tube walls, which could lead to loss of material and affect the quality of the library.