I purchased some oligos and now I need to create the adapter duplex from those oligos. This requires an annealing reaction which is super simple to do. Basically you just mix your oligos together, heat them to 95C and then slowly cool the mixture. Nature takes care of all the leg work. I’ve done this reaction 3 different ways:
- Heat a cup of water to boiling in a microwave, then remove the water, place your annealing mix in the water (make sure the top is floating), and allow the water to cool on a lab top. Basically allow the water to cool to room temperature (RT).
- Put your mix into a heating block and heat that to 95C (or close to boiling). Once the mix has been heated, remove the block from the heating unit and place on a lab top to cool to RT.
- Put your annealing mix in a PCR machine which can control the temperature very specifically. Create a program that will: (1) heat the mix to 95C for about 5 minutes, (2) slowly lower the temperature to about RT or 4C or whatever cool temp you want, (3) hold at that low temp until you are ready to remove it and move on.
For today’s reaction I will be using option 3. My protocol for the experiment is below. Unfortunately there is no easy way to verify the annealing reaction is successful. Well that’s not 100% true. You can run an SDS-PAGE gel which has the ability to resolve very short DNA sequences, but I don’t really have the equipment for that right now. I do have a high resolution gel that I’ve been wanting to try. Hmmmm…
And here is a link to what my annealing buffer is: Annealing Buffer Recipe.