Annealing short oligos - How to anneal short oligos? (Jul/30/2005 )
I had complimentary oligos synthesized by a company for me. I am now trying to anneal them. I have seen several previous postings on annealing oligos, but my oligos are only 22bp long. Are there any special precautions I should take when trying to anneal these? What is the best way to go about doing this. Presently, my oligos are dissolved in distilled H2O...should i lypholize them first? Please help!!!
Thanks in advance,
Annealing oligos should be pretty easy. Take a look at this pape where you can find protocols and annealing buffer.
What I usually do is to put two oligos in 1X annealing buffer, place the tube in a beaker with water heated to 94C. Let the water slowly cool down to 37C within a hour period. It is ok if your oligos have been dissolved in water but you have to make sure they are in 1x final annealing buffer. After annealing, you can run a gel with ds-oligos and ss-oligo side by side and you will see a difference in imigration between them.
Thanks so much for the response...I was thinking about running a gel, but wasnt sure which type of gel to run. My oligos are only 22bp long, so i dont think an agarose gel would work. Suggestions? Thanks!
Run a 4% agrose gel. use a 25bp DNA ladder has your marker. it will work.
A Metaphor or Nusieve 1:3 gel will work much better for these short fragments than a conventional 4% agarose gel. The conventional gel might work, but with relatively poor resolution.
you can alternatively run a non denaturing 15%polyacrylamide gel and color it by ethidium bromide, revealing your oligos.
I am thinking of running a 15% acrylamide gel...could someone send me a recipe? I hav a 19:1 acrylamide/bisacrylamide stock solution...A recipe using this solution would be greatly appreciated!!!
i use 19:1 or 29:1 without any difference.
Here is :
for 50ml final :
18.8 ml Acryl / Bis Acrylm
5ml TBE 5X
Pre run at 300v minimum 15'
Run at 200v with O.5 TBE buffer
After that : coloring in TBE 0.5X / BET (volume of solution = 5times Gel volume and [BET] final at 0,1µg / ml)
15' decoloration in TBE 0.5x
Take a picture and smile