help needed!

[Guest enghuei]

Describe how you would make 1 liter of a sterile 1 M solution of Tris, pH7, to be used for RNA isolation (molecular weight of Tris = 121 g/mol; pKD = 8.1)

 

anyone knows how to answer this?

[JaKiri]

Dissolve 121g of Tris into 1 litre of water, surely.

[Radical Edward]

don't you need something else for the pH?

 

I reckon you add a load of acid to it and then boil it for three weeks.

[JaKiri]

Radical Edward said in post # :

don't you need something else for the pH?

 

You'd better hope the neutralistation reaction produces something water soluable.

[wolfson]

Add (d&nr)H2O to 1 litre, autoclave for sterilisation that should be the solution, but I could do with knowing whether you are using the glacial acetic acid by-route step, if so use Na2EDTA by 3-2 TAE.

[VendingMenace]

You'd better hope the neutralistation reaction produces something water soluable.

 

It will. Tris is a buffer. ph 7 is well within its range. All you need to do is make the correct molarity solution of tris, then add either dilute HCl or dilute NaOH, until the desired pH is obtained.

 

Then, you can sterilize it either through autoclaving (not what i would do -- takes too long) or filtration (what i would do).

 

Cool

[YT2095]

VM, filtration to sterilise?

 

Autoclave is actualy the best way other than using chems (that may effect the product).

 

and HCl maynot be compatible either with the tris (whatever that is LOL).

if it`s for RNA (proteins) then NaOH would be out of the question, they`ll cook!

[JaKiri]

wolfson said in post # :

Add (d&nr)H2O to 1 litre, autoclave for sterilisation that should be the solution, but I could do with knowing whether you are using the glacial acetic acid by-route step, if so use Na2EDTA by 3-2 TAE.

 

Add it to water, not the other way round.

[VendingMenace]

VM, filtration to sterilise?

 

Yeppers

 

If the pore size is small enough then you are assured that it is steril. (kinda like how a backpack pump will work -- though that may be reverse osmosis is some cases)

 

Autoclave is actualy the best way other than using chems (that may effect the product).

 

Autoclave can work, but you have to be careful. Depending on what you are doing, the heat of the autoclave can cause your chemicals to deteriorate. Also, autoclave can take quite awhile, as you have to wait for the machine to run and the wait for the solution to cool. WIth 1 liter of water, this can take awhile. Filtration will be faster -- and in my opinion better, though more expensive.

 

and HCl maynot be compatible either with the tris (whatever that is LOL).

 

It is. And really you will not need to add much HCL to get the pH to where you want it. TRIS is a compound that is used particularely in biochemistry. It is used as a pH buffer when working with protiens, DNA, and the like.

 

if it`s for RNA (proteins) then NaOH would be out of the question, they`ll cook!

 

Not really. You make the buffer first, and then add the chemicals that you are studying. The buffer exists in order to keep the solution at the correct pH so that proteins and DNA do not denature. Thus, even though you are adding base, you are doing so only to bring the pH into the acceptable range for your experiment. Once you have done this, the pH is quite stable. So quite the oppsosite is true, adding NaOH to adjust the pH will, in the end, make your protiens more stable in the solution.

 

Though, without the buffer, your accertion would be correct

 

 

Add it to water, not the other way round.

 

Actually, it is correct to add water to the tris. It is incorrect to meauser out 1 liter of water and then add tris to it. The reason is that a solid, when dissolved will often occupy a different volume than when it was solid. Thus, you need to add the apporpraite amount of tris ot a 1 liter volumetric flask and then fill the flask to the full line. This is the way it must be done if you are to be assured that you have really made the correct concentration of tris

[Cookie]

 

 

 

Actually, it is correct to add water to the tris. It is incorrect to meauser out 1 liter of water and then add tris to it. The reason is that a solid, when dissolved will often occupy a different volume than when it was solid. Thus, you need to add the apporpraite amount of tris ot a 1 liter volumetric flask and then fill the flask to the full line. This is the way it must be done if you are to be assured that you have really made the correct concentration of tris [/b]

 

Plus, you want to leave room (volume wise, I mean) to adjust the pH with HCl. Then you fill to the mark with water.

 

Cookie

[chemistry]

pKD...never heard of it. pKa or pKb I can understand. If you had just made a typing error and pKD=pKa for example then...

 

Add water to 121g of the substance then add water until it reaches 1 L.

 

Find a buffer system with a pH of 7 and/or use the Henderson-Hasselbach equation or neutralize the substance with the proper acid or base. The latter will probably be easier.

 

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[wolfson]

The way is to Autoclave (have not said it for fun), like i said to reach a suitable pH then use the glacial acetic acid by-route step, with the addition of Na2EDTA. If you follow my steps you should create what you are intending to, and filtration is out of the question.

[YT2095]

I was actualy thinking about autoclaving (is there such a word? LOL) the water 1`st and then doing the mixing,

(it`s not my area so you`ll have to fogive me a little here).

 

I`ve always considered NaOH to be destructive to proteins (certainly fats).

 

what exactly is TRIS and it`s uses, it sounds fascinating

[wolfson]

Yes autoclaving is a word lol, tris is a Electrode Buffer, Stacking Gel Buffer and RNA isolator for use in Bio, and you are right about NaOH.

[YT2095]

an electrode buffer, are we talking electrophorisis here?

the gell stuff?

a bit like chromatography using a gell medium and an applied voltage to move the molecules into bands or regions through this gell?

 

cheerz

[wolfson]

Yep its a method of separating substances, especially proteins, and analyzing molecular structure using an eletric field, you hit thespot

[YT2095]

Darn! and I just drank all the Ninhydrin LOL

 

thanx yo

[wolfson]

Well i dont think i can get internal finger prints LOL

 

yw

[YT2095]

I should Jolly well hope not either! LOL

 

just on spec though, I have tried (to limited success) electrophorisis experiments here in the lab also, what electrodes are used, I used Carbon, it seemed to work best but there is still discoloration, I assume it`s platinum, but am uncertain (and don`t have much left).

[wolfson]

The electrophoresis electrodes are made of platinum wires

[YT2095]

I tried that but my wire was too thin

 

ya know these lighters that make a blue flame through a mesh, supossed to be impossible to blow out, well that silver wire mesh is made of Platinum

sadly it also becomes quite brittle and unmanagable over time, and certainly too thin to be a viable option

 

not to worry though, Live and learn eh

[VendingMenace]

and filtration is out of the question.

 

Care to explain why? I gave a reason why i would filter instead of autoclaving and i said why filtration will work. Now, why would you not filter??

 

I`ve always considered NaOH to be destructive to proteins (certainly fats).

 

To some extent you are corrret. Of course NaOH is only a stong base -- so the extent to which it damages protiens (denatures then) is only a funtion of its concentration. (Protiens denature at high pH's)

 

So some things to consider are;

1) You will not be adding much base to the solution

2) What you add will be soaked up by the buffer and you will end up with a pH of 7. This pH will not denature protiens.

 

You see, it really only matters what the pH is in solution. there is nothing magical about NaOH, any base will denature protiens, assuming that it can move the pH high enough...

 

 

Yep its a method of separating substances, especially proteins, and analyzing molecular structure using an eletric field

 

Electrophorisis tells us very little stuctural information by itself. It can at best differntiate between coiled, cut, and supercoiled DNA and RNA. WHen it comes to protiens you would be hopelessly lost when it comes to structural information.

 

Electrophorises is really just another size exclution colomn (one that takes charge into account in a limited sense) that is turned on its side.

 

In the end, all that electrophorises really tells you is your charge to mass ratio (and sometimes a very little bit of structural information.

[YT2095]

re: the 1`st part of your post that was echoed in your post #9.

no one mentioned filtering after that?

 

ph & is quite fine, how do you compensate for local denaturing though as you introduce this NaOH?

 

as for the rest of it, as stated, It`s not my area, and so sure I shall ask questions out of interest

[VendingMenace]

ph & is quite fine, how do you compensate for local denaturing though as you introduce this NaOH?

 

Ah, i see the source of confusion now

 

First you make the solution and adjust the pH and sterilize it, then you add the element of interest (protiens or DNA or whatever. So the thing you are studying is not in the solution when you are adding NaOH

 

Cool

 

as for the rest of it, as stated, It`s not my area, and so sure I shall ask questions out of interest

 

yeah, that makes sense. I am sorry if you thought i was getting furstrated? with you. I was merely trying to add my own thoughts to wolfson's

[YT2095]

no worries man

the "&" sign meant to be "7" in case anyone wondered, I know VM got it.

 

I am however curius abot this filter though? what exactly is it and how long would it take to pass a litre, assuminy by strilisation it means the removal of any Virus or Bacterial contaminats, that`s got be a pretty fine mesh! and take ages to do I should imagine, even with a vacuum pump!

 

I imagine it has activated charcoal and stuff though, any chance of a few pics?