Questions about DNA dialysis

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Questions about DNA dialysis

Postby spark » Jan 09 2013 6:33 am

Dear all,
I would like to use dialysis to exchange ions in my DNA. For this I bought Slide-A-Lyzer mini pots for the dialysis.
First, I am looking for more information about the theoretical background of dialysis. Can you give me some literature hints, please?

Secondly, I know my target DNA concentration, but I know dialysis can lead to dilution and sample loss. How much higher shall I choose the starting concentration?
Also, how can I calculate the optimum time for dialysis, the right temperature, please?

Thank you very much for your hints.
Kind regards
Spark

PS: I am a real newbie in this field, so sorry if my questions are super stupid.
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Re: Questions about DNA dialysis

Postby mchlbrmn » Jan 09 2013 5:25 pm

Sample loss would be a technical thing about he gizmo you bought. If you are not near the size cut off there shouldn't be much loss, I'd think, except the bit of volume stuck in the gizmo if you have small volumes, and perhaps a tad stuck in the pores.
It should work by simply allowing the ions to diffuse across the membrane so that the initial volume becomes the final volume, and the salts are diluted proportionately. Like, V1/V2 * inititial salt conc.
If you want to, rather than calculate dilution, you can just dialyze in large volume, or do several sequential dialysese (how do you pluralize?) to get close to nothing, then add salts/ingredients to desired concentrations.
I used to do larger volumes of DNA in large dialysis bag overnight in large flask, stirring, in cold room. Warmer is faster. A bit of Tris/EDTA as a dilution buffer is normal to keep DNA safe (5-10 mM Tris (desired pH, 8.0 is common), 0.1-1 mM EDTA (depending on whether downstream applications require Mg that EDTA removes.) I'm not sure how long it takes, but for my large bag it was hours, or overnight to wait for the diffusion to equilibrate the solutions (or to taste).
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Re: Questions about DNA dialysis

Postby spark » Jan 10 2013 10:03 am

mchlbrmn wrote:Sample loss would be a technical thing about he gizmo you bought. If you are not near the size cut off there shouldn't be much loss, I'd think, except the bit of volume stuck in the gizmo if you have small volumes, and perhaps a tad stuck in the pores.

Yes, I have DNA of weight around 15kDa. I chose a cut off of around 3kDa hoping not to loose sample. I am aware of the linear form of DNA.

mchlbrmn wrote:It should work by simply allowing the ions to diffuse across the membrane so that the initial volume becomes the final volume, and the salts are diluted proportionately. Like, V1/V2 * inititial salt conc.

You assume this simple equation because water should not be able to go through the membrane, correct?
What is V1 and V2?
Sorry, Chemistry is not my strength.

mchlbrmn wrote:If you want to, rather than calculate dilution, you can just dialyze in large volume, or do several sequential dialysese (how do you pluralize?) to get close to nothing, then add salts/ingredients to desired concentrations.

I thought anyway to repeat the dialysis because I have to remove certain ions and bring new ones in. I thought DNA needs charges. What does happen when you remove all ions without offering a replacement?
I need to avoid aggregation in case this happends when no salt is present.

mchlbrmn wrote:I used to do larger volumes of DNA in large dialysis bag overnight in large flask, stirring, in cold room. Warmer is faster. A bit of Tris/EDTA as a dilution buffer is normal to keep DNA safe (5-10 mM Tris (desired pH, 8.0 is common), 0.1-1 mM EDTA (depending on whether downstream applications require Mg that EDTA removes.) I'm not sure how long it takes, but for my large bag it was hours, or overnight to wait for the diffusion to equilibrate the solutions (or to taste).


Sorry, I have just a couple of uls. Why do you prefer cold although warm would be faster? Why do I need please Tris buffer? Would I need EDTA?
When I managed to exchange the ions and get a specific concentration, I am ready for the measurement. No other process shall be followed.
How did you know once you were finished with the dialysis, please?

Many thanks for this exchange.
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Re: Questions about DNA dialysis

Postby spark » Jan 10 2013 11:24 am

Dear mchlbrmn,
Thank you for your answer.

mchlbrmn wrote:Sample loss would be a technical thing about he gizmo you bought. If you are not near the size cut off there shouldn't be much loss, I'd think, except the bit of volume stuck in the gizmo if you have small volumes, and perhaps a tad stuck in the pores.

I chose a very low cut off, so the DNA does hopefully not escape. I think my DNA is 15kDa (very short) and I chose 2kDa and 3.5kDa as cut off.

mchlbrmn wrote:It should work by simply allowing the ions to diffuse across the membrane so that the initial volume becomes the final volume, and the salts are diluted proportionately. Like, V1/V2 * inititial salt conc.

Why, please? Because you assume no water exchange?
What is V1 and V2, please?

mchlbrmn wrote:If you want to, rather than calculate dilution, you can just dialyze in large volume, or do several sequential dialysese (how do you pluralize?) to get close to nothing, then add salts/ingredients to desired concentrations.

Yes, in fact, the aim is to completely exchange some ions. As I am aware I will run into an equilibrium, I aim for repeating the dialysis to exchange as much as possible.
Is there no danger to dilute the DNA during this process, please?


mchlbrmn wrote:I used to do larger volumes of DNA in large dialysis bag overnight in large flask, stirring, in cold room. Warmer is faster. A bit of Tris/EDTA as a dilution buffer is normal to keep DNA safe (5-10 mM Tris (desired pH, 8.0 is common), 0.1-1 mM EDTA (depending on whether downstream applications require Mg that EDTA removes.) I'm not sure how long it takes, but for my large bag it was hours, or overnight to wait for the diffusion to equilibrate the solutions (or to taste).


Sorry, I just got several ul and I think at my rather high concentration this will result only in a couple of ul.
Why is cooler better?
Why is Tris necessary to make it save?
I don't want to get out first all ions, because I am afraid the DNA will aggregate.
I just want to perform the ion exchange and measure it. No other processes shall follow. Do I need than EDTA despite?
When do you know it is finished, please?

Best
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Re: Questions about DNA dialysis

Postby mchlbrmn » Jan 10 2013 1:19 pm

Why, please? Because you assume no water exchange?
What is V1 and V2, please?
I always could be missing something, but I just mean it's like a simple dilution, with water and everything below the cut off diffusing across the membrane. V=volume. So, with 100ul dialyzed in a 1L beaker, it would be a simple 1/10,000 dilution of the salts and buffer concentrations.

Is there no danger to dilute the DNA during this process, please?
DNA is rather stable in general. It's normal to handle DNA in TE solution to be safe, as I sort of mentioned. Originally TE was Tris(pH8, or whatever you want downstream) and EDTA at 1mM, originally, but you can reduce it to 0.1 mM if it will interfere with downstream applications, or not add it at all if you know no Mg, or other divalent cations, are present. Common DNAse I, that destroys DNA, requires Mg as acofactor. EDTA chelates, and removes from solution a more than equimolar amount of EDTA (around 1.5 or 2, I think, moles Mg removed by 1 mole EDTA). Unlike RNAse, DNAse is destroyed by heat and other treatments, so DNA is more stable than RNA. However, if left for long periods in a weak acid solution, it will degrade, so it is normally kept in some pH buffer solution, like Tris. Even distilled water with no buffer tends to become acidic. So, it's normal to put DNA in TE (Tris 5-10mM, EDTA 0.1-1mM).

Is there no danger to dilute the DNA during this process, please?
Unlike protein DNA does not mind being dilute. As said above, as long as it has the pH buffered and no Mg present. If you are talking about minute amounts of DNA, then there would be some risk of it sticking to glassware or perhaps some other substrates, but ug amounts are not a problem. It doesn't precipitate in water (all those phosphpate charges on the backbone make it polar and very soluble).

Why is cooler better?
To prevent degradation of the DNA if left overnight. However in TE room temp would be OK also.

Why is Tris necessary to make it save?
Mentioned above, Tris is a pH buffer to maintain the pH.

I don't want to get out first all ions, because I am afraid the DNA will aggregate.
Said above, it won't. It's inherently highly soluble in water.

I just want to perform the ion exchange and measure it. No other processes shall follow. Do I need than EDTA despite?
If it's in EDTA now, or downstream and you haven't added any magnesium, or you know it has none, then it shouldn't be susceptible to DNAse degradation, so you can probably skip the EDTA. You might put a very low 0.1 mM in the first dilution with low buffer, and use water at the end. People actually commonly store oligonucleotides in only water, although stored frozen when not in use. I think oligos are considered exceptionally stable because of the size. If degradation occurs at 1/1000 bp then a 1kb DNA will be rather broken up, but only one in 100 10bp oligos will be cleaved, so it will be 99% intact. The risk in unbuffered water is auto acidic lysis, the DNA itself and carbonic acid from CO2 making water acidic, however this takes a while so is not always an immediate problem. So go ahead and use water for oligos (I get long winded because I hate committing like that. Might be safer to use buffer until the end.... there I go).

When do you know it is finished, please?
I don't know. (Concise, for once). Not so long for only ul's.
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Re: Questions about DNA dialysis

Postby mdfenko » Jan 14 2013 12:21 pm

for reference information on dialysis (for protein research) you can go to this website: dialysis methods for protein research

for a more complete overview, download this tech note: dialysis: an overview

and for more specific information for devices, download this brochure:high-performance dialysis

these should answer many of your questions.
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Re: Questions about DNA dialysis

Postby spark » Jan 14 2013 12:24 pm

mchlbrmn wrote:I always could be missing something, but I just mean it's like a simple dilution, with water and everything below the cut off diffusing across the membrane. V=volume. So, with 100ul dialyzed in a 1L beaker, it would be a simple 1/10,000 dilution of the salts and buffer concentrations.

To be precise the factor would be 100ul/(100ul+1000ml), right? I know 100ul are rather small in comparison, but just to get it right.
I guess, V=volume, but I did not get the numbers referring to.

mchlbrmn wrote:DNA is rather stable in general. It's normal to handle DNA in TE solution to be safe, as I sort of mentioned. Originally TE was Tris(pH8, or whatever you want downstream) and EDTA at 1mM, originally, but you can reduce it to 0.1 mM if it will interfere with downstream applications, or not add it at all if you know no Mg, or other divalent cations, are present. Common DNAse I, that destroys DNA, requires Mg as acofactor. EDTA chelates, and removes from solution a more than equimolar amount of EDTA (around 1.5 or 2, I think, moles Mg removed by 1 mole EDTA). Unlike RNAse, DNAse is destroyed by heat and other treatments, so DNA is more stable than RNA. However, if left for long periods in a weak acid solution, it will degrade, so it is normally kept in some pH buffer solution, like Tris. Even distilled water with no buffer tends to become acidic. So, it's normal to put DNA in TE (Tris 5-10mM, EDTA 0.1-1mM).

But where would the DNAse come from, when I ordered synthesized DNA? It should not be in the sample? I see why now EDTA and Tris, what is your preferred combination in terms of concentration?

mchlbrmn wrote: Unlike protein DNA does not mind being dilute. As said above, as long as it has the pH buffered and no Mg present. If you are talking about minute amounts of DNA, then there would be some risk of it sticking to glassware or perhaps some other substrates, but ug amounts are not a problem. It doesn't precipitate in water (all those phosphpate charges on the backbone make it polar and very soluble).

Re: DNA diluted by dialysis. Is there not a risk that there is a transfer of water molecules into the dialysis compartment? Or when would this happen during a dialysis, please?
What are minute amounts, please?


I don't want to get out first all ions, because I am afraid the DNA will aggregate.
mchlbrmn wrote:Said above, it won't. It's inherently highly soluble in water.

Even no risk of dilution inside the DNA compartment? I am a bit afraid that when I start with the dialysis, some gradient could be too steep, but maybe I am confused.

I just want to perform the ion exchange and measure it. No other processes shall follow. Do I need than EDTA despite?
mchlbrmn wrote:If it's in EDTA now, or downstream and you haven't added any magnesium, or you know it has none, then it shouldn't be susceptible to DNAse degradation, so you can probably skip the EDTA. You might put a very low 0.1 mM in the first dilution with low buffer, and use water at the end. People actually commonly store oligonucleotides in only water, although stored frozen when not in use. I think oligos are considered exceptionally stable because of the size. If degradation occurs at 1/1000 bp then a 1kb DNA will be rather broken up, but only one in 100 10bp oligos will be cleaved, so it will be 99% intact. The risk in unbuffered water is auto acidic lysis, the DNA itself and carbonic acid from CO2 making water acidic, however this takes a while so is not always an immediate problem. So go ahead and use water for oligos (I get long winded because I hate committing like that. Might be safer to use buffer until the end.... there I go).


So, you prefer always TE buffer than rather Tris only? Why do you hate committing like that?
Yes, safer shall be fine.

Another question: How would I determine the extinction coefficient for usage with a Nanodrop (UV-Vis)? I read one should use A260, but no coefficient was given.
Thank you for the information exchange.
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Re: Questions about DNA dialysis

Postby mdfenko » Jan 14 2013 12:38 pm

spark wrote:So, you prefer always TE buffer than rather Tris only? Why do you hate committing like that?
Yes, safer shall be fine.

since you "know" there are no dnases present then just 10mM tris, pH 8, will be fine.

Another question: How would I determine the extinction coefficient for usage with a Nanodrop (UV-Vis)? I read one should use A260, but no coefficient was given.


the nanodrop program will do the calculation for you. the extinction coefficients are built in to the program.
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Re: Questions about DNA dialysis

Postby spark » Jan 15 2013 5:26 am

Dear all, thank you for your hints and help so far.
What Tris do you mean exactly, please? I think I once found two different once in the former lab, but we don't have it here, so I would need to order.
Sorry, I did not know that the Nanodrop has the values for DNA. I was just aware it knows it for Lysoszyme.
Thank you.
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Re: Questions about DNA dialysis

Postby mdfenko » Jan 15 2013 8:44 am

tris, when represented with a defined pH, is a buffer prepared either by the addition of hcl to tris base (or naoh to tris-hcl) or by mixing equimolar tris base and tris-hcl until the desired pH is attained.

the booklet you can download at this website will help you to understand buffers and their preparation (clicking the link should download the booklet).
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Re: Questions about DNA dialysis

Postby mchlbrmn » Jan 15 2013 2:03 pm

So, you prefer always TE buffer than rather Tris only? Why do you hate committing like that?
Yes, safer shall be fine.
Well, I didn't know you were working with manufactured oligos originally, although I guessed they were some kind of oligo when you mentioned the molecular weight. TE is the old fashioned safe DNA storage solution, but it is also common to just keep oligos in water, thawed when in use and frozen for storage. So, you're probably fine with just water, or low concentration Tris alone if they never had magnesium present, but I'd hate to give advice that could cause trouble if incubated overnight, and, for example, the dialysis membrane introduced a little magnesium. Also, I don't know if Tris or EDTA interfere with your application, so I would be guessing to give definite advice.

To be precise the factor would be 100ul/(100ul+1000ml)
Yes, unless your beaker contains 999.9ml to start.

But where would the DNAse come from, when I ordered synthesized DNA? It should not be in the sample? I see why now EDTA and Tris, what is your preferred combination in terms of concentration?
I don't know, you could spit DNA into it, I suppose. It's RNAse that's a big problem since it's almost impossible to destroy, so is present everywhere, but you're right, probably no DNAse present, and if so will now work withoug magnesium.
TE is commonly 10mM Tris, 1mM EDTA (or 0.1 mM EDTA if the EDTA would interfere with downstream applications), or no EDTA if the DNA has no DNAse or magnesium present.

"Minute" = low number of ng/ml or less, I suppose.

Re: DNA diluted by dialysis. Is there not a risk that there is a transfer of water molecules into the dialysis compartment? Or when would this happen during a dialysis, please?
Do you mean by osmosis? Osmosis is usually strong with a semipermeable membrane that permits water to pass, but not salts. Your salts should pass through, so unless a little osmosis occurs before the salt concentration is equilibrated, not much should occur. I don't know if DNA itself can cause osmosis(?). Maybe not at these concentrations, if so. I assume you'll check the volume after dialysis.

Is there a reason you're doin dialysis instead of precipitation, which is faster? Perhaps it's better at removing all salts, if that's important?
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Re: Questions about DNA dialysis

Postby spark » Jan 16 2013 8:58 am

mchlbrmn wrote:Well, I didn't know you were working with manufactured oligos originally, although I guessed they were some kind of oligo when you mentioned the molecular weight. TE is the old fashioned safe DNA storage solution, but it is also common to just keep oligos in water, thawed when in use and frozen for storage. So, you're probably fine with just water, or low concentration Tris alone if they never had magnesium present, but I'd hate to give advice that could cause trouble if incubated overnight, and, for example, the dialysis membrane introduced a little magnesium. Also, I don't know if Tris or EDTA interfere with your application, so I would be guessing to give definite advice.

I just found out that the dialysis pots have 5ppb Mg, but that can be dialysed out with 15min in water soaking. But I think I will opt for TE buffer! As you said: safer!
No, I don't think that Tris or EDTA will interfere with my measurement. It is just in the buffer.

To be precise the factor would be 100ul/(100ul+1000ml)
mchlbrmn wrote:Yes, unless your beaker contains 999.9ml to start.

Okay, but at least I have the calculations right :-)

Re: DNA diluted by dialysis. Is there not a risk that there is a transfer of water molecules into the dialysis compartment? Or when would this happen during a dialysis, please?
mchlbrmn wrote:Do you mean by osmosis? Osmosis is usually strong with a semipermeable membrane that permits water to pass, but not salts. Your salts should pass through, so unless a little osmosis occurs before the salt concentration is equilibrated, not much should occur. I don't know if DNA itself can cause osmosis(?). Maybe not at these concentrations, if so. I assume you'll check the volume after dialysis.

The membrane is after all as well permeable for water and I imagine that my sample pot has less water molecules inside than the large beaker, so in principle there should be as well a transfer of water. I thought this is why people leave space in their dialysis bags to accommodate for the extra water? And is this not the reason why after dialysis also the sample concentration is reduced?
Maybe I am wrong, but I am as well not an expert.
I found in one manual:
Water is such a small molecule that it is capable of passing through the pores of virtually all dialysis membranes. When dialyzing a high solute concentration against a dilute dialysis buffer, there will be a net movement of water (and possibly salts) into the dialysis unit through the membrane. Glycerol and some sugars are especially hygroscopic, and as rapidly as they diffuse across the membrane to reach equilibrium, they also significantly affect the osmosis of water across the membrane and so may cause a change in volume of the sample. Take care when dialyzing with large differences in glycerol or sugar concentration between sample and dialysis membrane.
Prevent this movement of water and consequent change in sample volume by dialyzing in a "stepwise" fashion, minimizing the difference in water concentration between sample and dialysis buffer at each stage in the dialysis process. For example, when processing a sample with very high solute concentration against a buffer with very low solute concentration, dialyze first against a fairly concentrated dialysis buffer. With each subsequent replacement of dialysis buffer, use a less concentrated buffer until the desired final buffer concentration is reached.

So, in principle I dialyse first against a higher salt concentration and then I dilute this concentration with each buffer exchange?

mchlbrmn wrote:Is there a reason you're doin dialysis instead of precipitation, which is faster? Perhaps it's better at removing all salts, if that's important?

I want to remove some sodium, but would like to replace it for other salts.
I think my supervisor said precipitation will cause the DNA to aggregate and this is something I should avoid by all means for the following measurement.
Thank you very much for your patience and this information exchange!
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Re: Questions about DNA dialysis

Postby spark » Jan 16 2013 9:22 am

mdfenko wrote:tris, when represented with a defined pH, is a buffer prepared either by the addition of hcl to tris base (or naoh to tris-hcl) or by mixing equimolar tris base and tris-hcl until the desired pH is attained.

the booklet you can download at this website will help you to understand buffers and their preparation (clicking the link should download the booklet).


HI mdfenko.
The booklet writes:
Tris-HCl Buffer, pH range 7.2 to 9.0
and mentions
Tris base, no pH range mentioned.

I am still confused. So I can use Tris-HCl adjust with NaOH (I assume 1M NaOH) or Tris base and add strong HCl, please?
Which Tris would you use to rich pH 8, please?
Thank you.
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Re: Questions about DNA dialysis

Postby mdfenko » Jan 16 2013 10:24 am

spark wrote:The booklet writes:
Tris-HCl Buffer, pH range 7.2 to 9.0
and mentions
Tris base, no pH range mentioned.

I am still confused. So I can use Tris-HCl adjust with NaOH (I assume 1M NaOH) or Tris base and add strong HCl, please?
Which Tris would you use to rich pH 8, please?
Thank you.

the book refers to tris-hcl buffer as a solution whose pH has been adjusted with hcl. this is not exactly the same as solid tris-hcl. solid tris-hcl is the acid component for use to prepare tris buffers by mixing with equimolar tris base.

tris is the buffer component. tris-hcl is an acid component, tris base is the base component. the pH range is the range of pH that tris will be an effective buffer. outside that range and it is not really a buffer (will not significantly resist change of pH).

when we prepare tris buffers, we usually take tris base for a specific molarity and volume, add some water, adjust the pH with hcl (start with higher concentrations then, as we near the desired pH, use progressively lower concentrations) taking care not to exceed the desired volume, bring to final volume with water.

the better way is to take equimolar base and acid tris components and mix until you reach the desired pH (there are charts which will tell you how much of each component, you will still need to adjust a little). this way final volume doesn't matter, as long as you have enough for your experiments, the concentration of the buffering component (tris) will remain the same during adjustment.

as for your question about sample dilution during dialysis, yes, water will diffuse into the dialysis bag and increase the volume. you can, however, minimize this by making the dialysis bag tight. we do this by introducing air into the bag until it is rigid before tying off (hard to explain, easy to show (in person)).

(sorry about the double post, i had trouble uploading the post and it went in twice. i can't delete the second one)
Last edited by mdfenko on Jan 16 2013 10:30 am, edited 1 time in total.
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Re: Questions about DNA dialysis

Postby mchlbrmn » Jan 16 2013 10:26 am

Osmosis occurs when there is a salt gradient across a membrane as water crosses the membrane to "try" to equalize the concentrations. This is classically with a semipermeable membrane that the salt cannot cross, so the salt concentration remains high on one side. In this case, although the salt can diffuse across the membrane, during the time it takes to do this there will be a higher concentration on one side, so water will tend to move in that direction, increasing the volume.

There may be details about exactly what you are doing, or details of how DNA behaves at some level that your supervisor knows more about than me, but my understanding in general is that DNA is a very highly water soluble molecule, and that it can be quantitatively precipitated and redissolved without appreciable loss. At very large sizes DNA becomes less soluble (many thousands of base pairs), or if there are contaminants present they can cause the DNA to be less soluble. If you are talking about a normal DNA oligonucleotide in a solution of water and the common simple salts or buffer, I wouldn't expect it to have any trouble completely redissolving after precipitation. However, if your procedure is extremely sensitive to salts, this could be an issue as DNA must be precipitated as a salt, with sodium acetate being used most often. However, ammonium acetate is also a common alternative. I do not know if your procedure is so sensitive that the amounts of salt carried over from precipitation would matter.

Regarding tris buffer, I thought I'd mention that the distinctions being discussed are mostly the means to the end. Tris is basic by itself, and one way or another the pH must be adjusted to the desired level, and difference would be a bit more or less additional salts added. For procedures very sensitive to Cl ions, it is sometimes pH'ed with acetic acid to make Tris-acetate instead of Tris-HCl.

Edit: I just saw mdfenko's post. Hmm, I also posted twice, and the deleted one. I'll now delete dfenko's duplicate.
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