hybridization (or renaturation ?) - (Sep/05/2005 )

Hi there. I need to do some hybridization (or renaturation, I am still confused what is the difference) of complementary oligos but I have some (probably stupid ) questions.
1. Do I need to use these nitrocellulose membranes? My idea was to just to mix two complementary strands.
2. Is there any oligo concentration dependence of hybridization?
3. How do I monitor when that hybridization occurred (i.e. is there any “real time” signal (maybe like absorbance) that I can monitor to see when the hybridization occurred.


Any other advice to poor physicist making his first steps in biology is welcomed .

Hi there,

answers interspersed

Hi there. I need to do some hybridization (or renaturation, I am still confused what is the difference) of complementary oligos but I have some (probably stupid ) questions.

Hybridisation generally refers to annealing two strands of nucleic acid that haven't been together before, but can also mean a certain procedure in which you use oligos or short DNA fragments to localize a complimentary DNA sequence. Re-naturing means to get two strands back together, which have been together before and have been denatured (generally by heat).

1. Do I need to use these nitrocellulose membranes? My idea was to just to mix two complementary strands.

I can see where your confusion with the hybridisation comes in here... the membranes are often used in the procedure to identify complementary strands. In your case these are not neccessary. Simply mix them together, heat them up and let them cool down according to this protocol:

Method:
1. Dissolve oligos in STE Buffer (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA). The presence of some salt is necessary for the oligos to hybridize. Dissolve each oligo at high concentration (1 - 10 OD260 units / 100 uL).
2. Mix two stands together in equal molar amounts. If you do not there will always be single stranded material left over.
3. Heat to 94oC and gradually cool. For many oligos this can be as simple as transferring to the bench-top at room temperature. For sequences with significant hairpin potential, a more gradual cooling/annealing step is beneficial; this is easily done by placing the oligos in a water bath or temp block and "unplugging the machine".
4. The resulting product will be in stable, double-stranded form and can be stored at 4oC or frozen.

Things to consider: If the product will be used in a ligation reaction, the addition of 5' -phosphate may be needed. This can be done at the time of oligo synthesis (chemical phosphorylation) or at any time thereafter using PNK (enzymatic phosphorylation). If the oligos are relatively long or to be used in cloning, starting with PAGE purified oligos is recommended.


2. Is there any oligo concentration dependence of hybridization?

You should have equal molecular ratio.

3. How do I monitor when that hybridization occurred (i.e. is there any “real time” signal (maybe like absorbance) that I can monitor to see when the hybridization occurred.

Only if you have especially modified (and very expensive) oligos. But really if your oligos aren't longer than 50bp and aren't prone to very extreme secondary structure formation, the above protocol will pretty much do it. You might be able to purify the annealed double-stranded oligo from a 20%TBE acrylamide gel, but thats loads of work and really not neccessary. DNA after all wants to stick together, otherwise our cells would have a little problem.


Any other advice to poor physicist making his first steps in biology is welcomed .

OK if you gonna stick with molecular biology you could do worse than buy (or get your hands on a lab copy) of "Molecular Cloning" also called Maniatis or Sambrook after the authors. Both amazon.com and Sigma sell it. In my opinion every lab should have one and if they did and people would actually read it, there wouldn't be a quarter of the questions posted here.
Another good resource is the protocols section of this website (button right on the top of this site).

Hope that helps

Oh and I forgot to mention, there aren't any stupid questions...

Hi there. I need to do some hybridization (or renaturation, I am still confused what is the difference) of complementary oligos but I have some (probably stupid  ) questions.
1. Do I need to use these nitrocellulose membranes? My idea was to just to mix two complementary strands.
2. Is there any oligo concentration dependence of hybridization?
3. How do I monitor when that hybridization occurred (i.e. is there any “real time” signal (maybe like absorbance) that I can monitor to see when the hybridization occurred.


Any other advice to poor physicist making his first steps in biology is welcomed  .

Thank you so much LeserattePD. May the force be with you .

May I also recommend the book "At the Bench" by Kathy Barker, for its excellent practical advice in surviving your first few months doing molecular biology.