Hi. We isolated genomic DNA using the phenol-chloroform method and stored it at -80°C for approximately 15 years. Currently, we would like to use this DNA for exome sequencing analysis. Given these conditions, would the stored DNA be considered suitable for NGS applications? Are there any additional steps you would recommend to ensure its quality and integrity for sequencing?
yes phenol-chloroform method DNA extraction method can be used if going for exome sequencing. but DNA quality and quantity should not be compromised.
Seda Nur Homurlu Good afternoon!
That’s a very long storage period. You could certainly try using it, but I recommend checking the DNA concentration before sequencing. Additionally, it might be a good idea to clean the DNA to ensure optimal results.
What sort of NGS are you using? If you are hoping for long-reads, then length/degradation will matter more than if you want shorter.
Check the concentration & quality using either nano drop + gel or use a Qbit
Hello Anzhelika Yudinceva than you for your reply. How can I clean DNA? Are there any kit for this purpose or what method would be appropriate?
Hello Katie A S Burnette thank you for the clarification. We are going to use 150 bp paired-end reads with 100x coverage. Are this conditions appropriate?
Hello Prem Kumar Meher thank you for the answer. Would you recommend any additional steps to ensure complete removal of phenol residues from the samples?
Repeating the isopropanol wash can help ensure the complete removal of any remaining phenol
Good day, Seda Nur Homurlu ! Column purification is commonly used, but it may not always be necessary. Since your DNA sample is valuable, I recommend contacting the sequencing center directly to clarify their requirements. Often, these facilities assess DNA quality and can handle sample preparation themselves. To avoid any risk of sample degradation, explain your situation to the technicians - they can optimize the process based on their methods and equipment.
- Badges
- Science method