Hello backers! Thanks to all of you I am back in the lab processing my samples. I have 15 sets of samples to prepare for sequencing, and as I plan on comparing them to each other, one of my top priorities in ensuring that no cross-contamination occurs between them during the processing. This means is I have to work with each set individually and then systematically clean all of my equipment before starting the next set. My current work flow is:
1: Finishing cleaning the viral DNA from the samples
2: Convert the single-stranded DNA to double-stranded DNA via amplification
3: Determine the amount of DNA present, dilute it to the proper amount, and chop it up into small pieces
4: Prepare the fragmented DNA for sequencing
Yesterday I was able to finish cleaning 6 sets of samples, and right now I have one of those sets amplifying! Steps 1 & 2 will go rather quickly and should be completed for all the samples within the next couple of weeks. Steps 3 & 4 will be completed together for each sample and will take 1 full day per sample to complete. As steps 3 & 4 are harder to schedule in around my other commitments they will probably take 5 weeks to complete for all 15 samples. Therefore, by the end of May I can hopefully have all of the samples processed and ready for sequencing!!!
Throughout this process I will periodically update you on my progress and send you new pictures. I hope you all have a fantastic rest of your week and weekend!!!
Here are the boxes with all my samples nicely frozen away at -20 degrees celsius, which is equivalent to -4 degrees Fahrenheit! We keep them well below the freezing point of water to keep the samples stable for long-term storage.
Above is one set of samples in what is called a speedvac. A lid closes down over them and then the samples spin while a vacuum dries any liquid from them. The spinning keeps the DNA at the bottom of the tubes while the vacuum slowly removes the liquid. This allows me to re-suspend the DNA in a specific amount of water.
This machine is a thermocycler. It rapidly heats and cools the samples to specific temperatures and keeps them at that temperature for a set amount of time, which is required for multiple steps of my processing. The set of samples I amplified today were inside the machine while I took the picture, as the amplification occurs at one temperature for 90 minutes and then the machine quickly raises the temperature to stop the reaction and then cools them to refrigerator temperature until they are removed.
These are the tubes with the samples I amplified today. The penny is there so you can see what a small volume of liquid it is that I am working with. The blue rack behind the tubes is what holds the tubes while I add and remove liquid from them. After the next step tubes 1-6 will be combined for the final preparation and sequencing. The tube with the plus sign at the far right hand side is what we call a positive control, this has a standard amount of DNA in it that was provided by the company who makes the amplification kit and is used to test against in case anything goes wrong with the amplification of my actual samples.