I'm regularly doing long pre-synthesized inserts of several hundred bp if I have to create a de novo sequence and what Kevin mentioned is working well. Just some additions:

1. Make sure the oligos are properly desalted, check with the company what protocol they use. Try another if you keep having degradation like you describe. Some companies do HPLC standard for desalting which works really well, I know Thermo Fisher does for their oligos, very good quality from my experience.

2. If your oligos are above 50-60 nt the percentage of properly full length synthesized oligos will be decreasing rapidly. That means you don't see degradation, but you see premature synthesis termination with very low percentages of full length oligos. So ideally you have filtered them first for proper length through PAGE. You can do that yourself or you order them with PAGE filtering, again Thermo is doing that for example. I think the price per Oligo was $35 extra (try it on your own and you know how much work it is and how much you are paid to estimate if it is worth it).

3. If you decide to go with the short-overlapping-oligo system which I can really recommend (use max 50 nt and you should be fine without HPLC and PAGE) then phosphorylate the oligos prior to annealing. For annealing I use a buffer which has final 10 mM Tris pH 7.4, 1 mM MgCl2, 100 mM NaCl. Make sure you have your oligos not (!) solved in TE buffer, the EDTA will basically chelate the magnesium ions and interfere with the proper folding/annealing ...! I know a lot of people believe Tris-EDTA will help to buffer the water and thus prevent degradation but ... honestly, DNA and RNA too have such insane charge on such a tiny spatial area, no EDTA or water with slightly acidic pH will be able to do anything there. If unsure, measure the pH of your water, anything that is at least pH 6 I would use without any worries as is and do for years for DNA and RNA, even stuff with many freeze-thaw cycles. Way more important is desalting for stability.

4. Profit.

No, it could be that the oligo itself has a tricky 3d-structure and uppon annealing with the other oligo this is destroyed making it migrate more. Did you try to run it under denatured conditions to compare structural effects?

Hello, thanks for helping!

No, I only ran the ssDNA (Forward/reverse) in water versus the dsDNA after the annealing reaction (the oligos were commercially synthetized and I need to anneal them to produce a fragment for cloning).

Eduardo,

You should heat them at 80 oC or so and then let them cool slowly at room temperature. You can use the PCR machine and a stepwise temperature program until you reach room temperature or even down to 4 `C.. Report back

After the annealing, when they have reach the room temp you can directly use for ligation with your linearized vector. Some timeit is possible to obtain strange results running oligos on gels but the annealed oligos can be good enough for ligation. Use different concentration of the annealed oligos in the ligation mix

Good luck

You shoud use agarose (at least 3%; 1% is quite inappropriate) or polyacrylamide gel; try denaturing conditions to assess any structural or degradative effect.

If you repeat the annealing procedure, heat oligo mix in a thermal cycler up to 80°C for 2-5 minutes to minimize degradative effects. Use the same buffer and try to supplement the annealing buffer with 10% DMSO or formamide (at least 1M betaine should work as well).

Good luck

Sorry, I didn’t read all your thread. You should run the annealing mixture at 2% agarose or better a native PAGE 5% acrylamide. The dss oligo will be brighter under the UV. The smear could result from overloading. Maybe you could post a gel image.

I also agree with Anna. You can proceed to cloning since only productive annealing will ligate

Did you check for multiple complementary sequences within your oligo? I suspect that you could be getting multimers, which may not be easy to fix. DMSO is commonly used to PCR amplify GC rich sequences, and I wonder if it would be of any help here?

I agree with previous posts that heating to ~90C and cooling on the bench top is usually good enough. The step-wise thermocycler method maybe be a bit too much here because holding each temperature for a minute will allow for non-specific annealing, which may be contributing to your smear. Although not always, some times in science the simplest method is the best method.

Hi,

I have used the Mycobacterial High GC 85 bp long custum complementary oligos for cloning. they were designed to generate the overhang at the ends for cloning purpose, may be same as you are doing. I have done acconding to sigma annealing buffer composition (http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/annealing-oligos.html) and followed the protocol in waterbath (1.5 liter volume). I boiled the water on cooking heating plate and after boiling, I removed the container and kept aside withoud disturbing it. it worked nicely. If you are getting smear.. then try to first heat the buffer til 70C and then add the oligos. Boil for just 5 min and then take off from the heater.

It is difficult to prevent non-somplementary binding of primers to give just 80bp dsDNA using thermocycler. The >80bp smear is most likely primers primers not bound in the correct way. If there are some restriction sites engineered onto both ends of your 80bp DNA, use them and to clone it in a plasmid, and during colony screening in Ecoli, look for a colony with plasmid which give PCR product corresponding to an 80bp insert. Hope it works out for you.

Hi all,

To obtain a long double stranded DNA with sticky ends, I started from multiple oligos (see attached file) instead of only 2 long oligos in order to avoid having internal annealing and secondary structure. Mix the oligos in PBS1X and put the tube in a water bath. Boil the water for 5min and let the water cool-down to room temperature.

Hope this help,

Cheers

something a bit similar as previous post.

I design 2 oligos with an 18-25 bp overlap which will anneal (you can use high temperature), then I use klenow to fill up and restriction digest to make sticky end.

I agree with Genevieve, though Taq polymerase a single cycle, 96C for 5 mins and 72 for 20 mins will ok as well

Dear Eduardo,

I assume that you are going to use the annealed product as an adaptor or a linker of some sort for downstream application. I used to prepare a linker/adaptor for my thesis work which worked perfectly for me. You have to understand that annealing may not work at 100% efficiency. So you will always end up with some degree of ssDNA contamination in your prep. If you are planning on ligating the product to another DNA molecule using the sticky end, you would want the ligating end to be phosphorylated as well depending on whether the DNA to be ligated to has phosphate group or not. If you phosphorylate, then it is easy to check for annealing integrity by ligating the annealed product to itself so that you end up with 160 bp of ligated product versus 80 bp mostly ssDNA. The linkers will not ligate unless they are annealed dsDNA. It is going to be difficult to distinguish between 80 bp annealed dsDNA product and an 80 base ssDNA on agarose gels. I am willing to share my protocol with you if you are interested. If you are looking at only annealing the oligos, any PCR buffer that claim to be good for high GC content PCR (e.g, Qiagen's kit with Q-solution) should be good enough. You may do the annealing on a PCR block by heating the mixture to 95 C and cooling slowly @ 1C per minute to 25 C. I suggest thermal cycler in order to control the cooling rate. Otherwise as many people have suggested, incubating the tube in a container with water at 95 C and allowing it to sit at room temp until it cools down to RT should work equally well.

I'm regularly doing long pre-synthesized inserts of several hundred bp if I have to create a de novo sequence and what Kevin mentioned is working well. Just some additions:

1. Make sure the oligos are properly desalted, check with the company what protocol they use. Try another if you keep having degradation like you describe. Some companies do HPLC standard for desalting which works really well, I know Thermo Fisher does for their oligos, very good quality from my experience.

2. If your oligos are above 50-60 nt the percentage of properly full length synthesized oligos will be decreasing rapidly. That means you don't see degradation, but you see premature synthesis termination with very low percentages of full length oligos. So ideally you have filtered them first for proper length through PAGE. You can do that yourself or you order them with PAGE filtering, again Thermo is doing that for example. I think the price per Oligo was $35 extra (try it on your own and you know how much work it is and how much you are paid to estimate if it is worth it).

3. If you decide to go with the short-overlapping-oligo system which I can really recommend (use max 50 nt and you should be fine without HPLC and PAGE) then phosphorylate the oligos prior to annealing. For annealing I use a buffer which has final 10 mM Tris pH 7.4, 1 mM MgCl2, 100 mM NaCl. Make sure you have your oligos not (!) solved in TE buffer, the EDTA will basically chelate the magnesium ions and interfere with the proper folding/annealing ...! I know a lot of people believe Tris-EDTA will help to buffer the water and thus prevent degradation but ... honestly, DNA and RNA too have such insane charge on such a tiny spatial area, no EDTA or water with slightly acidic pH will be able to do anything there. If unsure, measure the pH of your water, anything that is at least pH 6 I would use without any worries as is and do for years for DNA and RNA, even stuff with many freeze-thaw cycles. Way more important is desalting for stability.

4. Profit.

Hello everyone, thanks so much for helping.

My oligos have high Tmelting, hence using 97ºC. It makes sense what some of you said about the smear corresponding to unproper annealing. I repeated the annealing and it improved a little bit but because the annealing will produce sticky ends that should in theory fit perfectly the sticky ends on the other fragments, I will just use a high amount of this linker and hope that whatever was able to anneal proper will be ligated while all the rest should be excluded from ligation. Will get back to you if whatever suggestions work out for me!

Thanks again

Hi there, we regularly make deletion cassettes for BAC recombineering using long overlapping oligos (100nt). This is what we do:

Oligo F (500uM) 2ul

Oligo R (500uM) 2ul

ddH2O. 6ul

Denature at 95C for five min

Transfer to a 37C water bath and incubate 30 min

We then run the annealed oligos through polyacrylamide mini gels and isolate the highest (and strongest) band.

Hope it helps.

I usually resuspend the oligos in water at high concentration (I always resuspend 1 in 50µl and the second in how much will give an equimolar concentration, I don't bother with the specific concentration as long as both oligos are the same) I then add 1µl of each oligo to 48µl of annealing buffer (100 mM potassium acetate,30 mM HEPES-KOH pH 7.4,2 mM Mg-acetate) and in a PCR machine 4 minutes at 95ºC,10 minutes at 70ºC and Slowly cool down to 4ºC.

A few comments.

First, 1% agarose is not the right gel to see the difference between 80 bp DNA fragments. you can increase the agarose to >2%, but I would just go to acrylamide gels instead.

Second, running single stranded oligos, is a misnomer. Without seeing your sequence I can still assume such long DNA strands will likely pair up randomly as well as having various forms of self-hairpins. You may be seeing aggregates of "ssDNA" that run larger than the annealed sample. You could try denaturing them at 95C+ then directly putting onto ice before running on a gel.

Ulrike,

I basically agree with most of what you have said but with these discrepancies.

1/ The EDTA is added to Chelate mostly zinc ion from nucleases that may contaminate DNA preparations. Almost all known nucleases have a zinc ion in their catalytic center.

2/ To check the purity of long oligos and purify them it is better to do it through a denaturing Urea gel or a mini sequencing gel.

3/ I am not sure I have heard about any known chemistry that implicates salt in the degradation of DNA or RNA.

Hi Ulrike, I wanted to also ask a similar question. My oligos are 63 bp long each. After reading this thread, I got them PAGE purified. But the yield became less. Can you tell me the procedure to anneal these oligos like how much should be the stock concentration while resuspending the oligos? What should be the working concentration to anneal them and in how much buffer volume? As I want to proceed with ligation of the ds oligo with a vector, how much should I dilute the dsoligo to ligate with the vector and finally what should be the insert:vector ratio? I would be highly grateful for any help.

Hello everyone!

Thanks again for tying to help me out with this. In the end the problem was solved by designing small primers to anneal on the big oligos and thus amplify the whole region of 80 bp. It worked!

Thank you so much for all your comments, hope I can help you people in the future somehow.

Hello, I will do the same experiment and I have a question! The 2 oligos need to be 5-phosphorylated prior ligation? I think they only have to be 5-phosphorylated if the vector is dephosphorylated, right?

Kyriakos Hassapis: yes, at least either the vector or the oligos need to be phosphorylated for ligation to happen.