@all It is unlikely that fish eggs fixed in 95% ethanol at room temperature for 1-2 years will be suitable for genetic barcoding. The DNA in the samples may have degraded over time, resulting in low quality or insufficient amounts of DNA for genetic analysis.

The quality of DNA can be affected by several factors, including the conditions under which the samples were stored and the length of time they were stored. Ethanol fixation can help preserve DNA, but it is not a guarantee of long-term preservation, especially if the samples were not stored at an appropriate temperature or for an extended period.

It is possible that some DNA may still be recoverable from the fish eggs, but the success of genetic barcoding will depend on the quality and quantity of DNA in the samples. To assess the suitability of the samples, it would be advisable to extract DNA and run a PCR test to determine if the DNA is of sufficient quality for genetic barcoding. However, given the extended period of storage, it may be more efficient to obtain fresh samples for analysis.

Hi Ahmad Al Khraisat, Thank you for the thorough answer. One additional questions if I may.

When you say PCR test, what exactly do you mean? Perhaps a RT-PCR? The reason I ask is because when I run PCR on them currently, there is sufficient quantity for sequencing (usually between 5-10 ng/uL), but I have no way of knowing the length of the amplicons of course. Would you advise running a gel-electrophoresis before I prep for sequencing to assess the suitability for sequencing?

Thank you.

Fish eggs fixed in 95% ethanol at room temperature for 1-2 years may still be suitable for genetic barcoding, but the quality and quantity of DNA may have degraded over time, which could affect the success of the barcoding experiment. The stability of DNA in ethanol depends on several factors, including the quality of the starting material, the concentration of ethanol, and the storage conditions.

When stored properly, DNA in ethanol is generally stable for long periods of time. However, prolonged storage can result in degradation, fragmentation, and other forms of damage that can impact the quality and quantity of the DNA. The quality and quantity of DNA in your samples can be assessed using methods such as gel electrophoresis, spectrophotometry, or fluorometry.

If the DNA is degraded or fragmented, it may still be possible to extract enough DNA for barcoding using specialized techniques such as PCR amplification or whole genome amplification. However, it is important to note that degraded DNA may not yield reliable or accurate results, and there may be limitations to what can be achieved with the available samples.

In summary, fish eggs fixed in 95% ethanol at room temperature for 1-2 years may still be suitable for genetic barcoding, but the quality and quantity of DNA may have degraded over time, which could affect the success of the experiment. It is recommended to assess the quality and quantity of DNA in the samples before proceeding with the barcoding experiment.

@all Paul Salib Hello! I apologize for any confusion caused. When I mention PCR test, I am indeed referring to the RT-PCR (Reverse Transcription Polymerase Chain Reaction) method, which is commonly used for amplifying RNA sequences. It is typically employed to detect and quantify specific RNA targets, such as viral RNA in the case of diagnosing infectious diseases.

Regarding your question about assessing the suitability of amplicons for sequencing, running a gel-electrophoresis can be a helpful step. Gel-electrophoresis allows you to visualize the amplified PCR products and determine their size range. By comparing the band sizes with a DNA ladder or marker of known sizes, you can estimate the length of your amplicons.

This information can be useful before proceeding with sequencing, as it helps to ensure that the amplified fragments are within the desired size range for your specific sequencing method. If the amplicons fall within the expected size range and have sufficient quantity, it increases the likelihood of obtaining successful sequencing results.

Therefore, running a gel-electrophoresis prior to sequencing can be a good practice to assess the suitability of your amplicons and ensure they meet the requirements for your sequencing experiment.