Understanding the basics of concentrations

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Understanding the basics of concentrations

Postby John Buckels » Jul 26 2006 7:46 am

I found this worksheet I used to use to train new starters in my old lab, and seeing as how there are so many questions on this topic I thought I would post it. Also included are some questions I used to give them - I wouldn't let them in the lab until they understood fully and got all the questions right


CALCULATION OF MOLAR, % AND "X" SOLUTIONS.

1. Molarity
A molar solution is one in which 1 litre of solution contains the number of grams equal to its molecular weight.

Example: To make up 100 ml of a 5M NaCl solution =
58.456 (mw of NaCl) g x 5 moles x 0.1 litre = 29.29 g in water to a final volume of 100 ml

2. Percent solutions.
Percentage (w/v) = weight (g) in 100 ml of solution;
Percentage (v/v) = volume (ml) in 100 ml of solution.

Example: To make a 0.7% solution of agarose in TBE buffer, weigh 0.7 g of agarose and bring up volume to 100 ml with TBE buffer.

3. Weight/Volume solutions.
This is the usual concentration for nucleic acids. A 1mg/ml solution contains 1mg of X per ml of solution.
Concentration = Weight
Volume

Example: To make 5 ml of a 200ug/ml solution of DNA =
5ml (volume) x 200ug/ml (conc.) = 1000ug of DNA (or 1mg)
If the stock DNA is at 10mg/ml =
1mg (weight needed) = 0.1ml (0r 100ul)
10mg/ml (conc.)

4. "X" Solutions.
Many enzyme buffers are prepared as concentrated solutions, e.g. 5X or 10X (five or ten times the concentration of the working solution) and are then diluted such that the final concentration of the buffer in the reaction is 1X.

Example: To set up a restriction digestion in 25 ul, one would add 2.5 ul of a 10X buffer, the other reaction components, and water to a final volume of 25 ul.

5. OD
When reading the absorbance of DNA using UV light at 260nm, in a 1cm path length, 1 OD unit is equal to 50ug/ml. Any dilution you have made can be incorporated into these calculations.

Example: 30ul of a DNA solution, diluted to 300ul with water, gives an OD reading of 0.437.
0.437 x 50 x 10 (Dilution factor = 300ul/30ul) = 218.5ug/ml



PREPARATION OF WORKING SOLUTIONS FROM CONCENTRATED STOCK SOLUTIONS

Many buffers in molecular biology require the same components but often in varying concentrations. To avoid having to make every buffer from scratch, it can be useful to prepare several concentrated stock solutions and dilute as needed.

The following is useful for calculating amounts of stock solution needed:
Ci x Vi = Cf x Vf

Where: Ci = initial concentration, or concentration of stock solution;
Vi = initial volume, or amount of stock solution needed
Cf = final concentration, or concentration of desired solution;
Vf = final volume, or volume of desired solution

Example: To make 100 ml of TE buffer (10 mM Tris, 1 mM EDTA), combine 1 ml of a 1 M Tris solution and 0.2 ml of 0.5 M EDTA and 98.8 ml sterile water.

Tris 1000mM x Vi = 10mM x 100ml so Vi = 1000/1000 = 1ml or 1000ul
EDTA 500mM x Vi = 1mM x 100ml so Vi = 100/500 = 0.2ml or 200ul


Conversion Factors
1x103g = 1kg (kilogram) = 1000g
1x10-3g = 1mg (milligram) = 0.001g
1x10-6g = 1ug (microgram) = 0.000001g
1x10-9g = 1ng (nanogram) = 0.000000001g
1x10-12g = 1pg (picogram) = 0.000000000001g
1x10-15g = 1fg (femtogram) = 0.000000000000001g


Questions

1. DNA Extraction necessitates the use of 70% ethanol. How do you prepare 1.5 litres of 70% Ethanol?

2. 1X Buffer K1 contains the following:
50mM NaCl, 25mM Tris-HCl, 10mM EDTA, 1% v/v SDS.
Calculate the amounts of each chemical required to make 250ml of 1X K1

3. Dale asks you to make 400ml of Buffer P1. In the Purple Lab you find bottles containing 0.5M solutions of Tris pH8.3, Potassium chloride and Magnesium Chloride as well as stock bottles of Tween 20 and Nonidet P40.
1X P1 contains 25mM Tris pH8.3, 50mM KCl, 2mM MgCl2, 0.45% v/v Tween 20, 0.45% v/v Nonidet P40
How do you go about making the buffer using the chemicals to hand?

4. How much Calcium Chloride is needed to make 80ml of a 12% w/v solution?

5. How much Decon is needed to make 500ml of a 2% v/v solution?

6. Dawn asks you to make 4 litres of 1X Buffer S3 (6M Ammonium Acetate). How much powder does this need?

7. You have isolated some DNA of unknown concentration. To quantify, you have taken 10ul of the DNA and diluted it to a final volume of 200ul. When read for absorbance at 260nm in a standard 1cm cuvette, this DNA gives an OD of 0.645. What is its concentration?

8. Your DNA from #7 needs to be diluted to 20ug/ml in a Master Plate. To make 100ul at this concentration, what volume of stock solution is required?

9. You need to aliquot 50ng of your DNA from the 20ug/ml Master Plate, for PCR. What volume do you require?

10. You need to run 200ng of your DNA on an agarose gel. What is the volume of 20ug/ml DNA required, and what volume of 5X sucrose loading buffer do you need to add to obtain the correct loading concentration? What would be the volume of 2X sucrose loading buffer required?

11. John has asked you to digest 3ul of PCR product with 3 units of EcoRV restriction enzyme. EcoRV is at a concentration of 20,000U/ml and requires reaction conditions of 1X NEBuffer 3 and 1X BSA. For a 10ul reaction, what volumes of Buffer, BSA, Enzyme, Water and PCR product are required?

Chemical Molecular Weight (fw)
Ammonium Acetate 77.08
Calcium Chloride 111.0
EDTA 380.17
Sodium Chloride 58.44
Tris 121.1


Questions - 2

1. How do you prepare 3.5 litres of 40% Glycerol from a 100% Glycerol stock?

2. Calculate the amounts of each chemical required to make 100ml of 5X KGB

3. How would you go about preparing a 1X solution of TBE from the 10X stock?

4. Describe what components you need to make a 100ml 0.8% Agarose gel

5. John asks you to make 2 litres of Buffer C1. In the Purple Lab you have bottles containing 1M Tris pH8.0 and 0.5M EDTA pH8.0 which you prepared earlier. How do you go about making the buffer using the chemicals to hand?

6. How much Silver Nitrate is needed to make 750ml of a 15% solution?

7. How much Silver Nitrate is needed to make 750ml of a 450mM solution?

8. You have isolated some DNA of unknown concentration. To quantify, take 30ul of the DNA and dilute it to a final volume of 600ul. What is the OD at 260 and 280nm? Work out the concentration of this DNA solution.

9. Your DNA from #8 needs to be diluted to 20ug/ml in a Master Plate. To make 100ul at this concentration, what volume of stock solution is required? How much TE is needed?

10. You need to aliquot 50ng of your DNA from the 20ug/ml Master Plate, for PCR. What volume do you require?
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Postby RS » Jul 26 2006 12:44 pm

A lot of supervisors need to take a lesson from you, that's for sure. I think that A LOT of time and energy is wasted by new students coming into a lab and supervisors expecting that they'll just sorta pick up things as they go along. Sure, we learn these things during undergrad, but it should be the supervisor's responsibility to ensure everyone knows what they're doing when they come into the lab. Honestly, I think the way many supervisors run their labs results in so much waste. Wake up science world!
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Postby John Buckels » Jul 26 2006 3:59 pm

I have tons more of stuff like this. Pipetting exercises (no-one seems to learn how to pipette at university so it saves a lot of hassle just to get them to learn and practice), reaction optimisations etc

I take pride in teaching my pupils :)
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Postby creepster » Jul 26 2006 4:23 pm

John Buckels wrote:I have tons more of stuff like this. Pipetting exercises (no-one seems to learn how to pipette at university so it saves a lot of hassle just to get them to learn and practice), reaction optimisations etc

I take pride in teaching my pupils :)


if you can share those documents (most of all the pipetting) i'd appreciate it
i tried similar exercises (like the mathematics stuff) but it seems that students cannot think nowadays
otherwise i'd suggest a personal visit and training session done by you
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Postby r.rosati » Jul 26 2006 5:02 pm

That's a very useful post John! Thanks for sharing it!!!

-Rob
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Postby John Buckels » Jul 28 2006 8:02 am

Maybe I should think about consultancy work.......

I'll dig out the other stuff over the weekend and see what I can find
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Postby plutoniah » Jul 30 2006 9:38 pm

very nice post, except that you may want to change your first molarity example, as 5M NaCl is not feasible at RT.
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Postby r.rosati » Jul 31 2006 1:06 am

plutoniah wrote:very nice post, except that you may want to change your first molarity example, as 5M NaCl is not feasible at RT.


Yes, it is feasible. Solubility of NaCl in water at 25°C is about 360 g/l, which is about 6.15M.
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Postby plutoniah » Jul 31 2006 3:34 am

no offense WeridOmen, but you may want to read through John's post. You're right that the solubility of NaCl is 360 g/l at RT, but that means that you can add 360 g NaCl to 1 L water. The final volume will be more than 1 L and thus the concentration will not be 6.15 M.

The actual solublity of NaCl at RT is 4.5 M. That's 264.6 g disolved in a final volume of 1 L. Even at 100 C, the solubility is only 4.8 M.
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Postby John Buckels » Jul 31 2006 7:31 am

plutoniah wrote:very nice post, except that you may want to change your first molarity example, as 5M NaCl is not feasible at RT.


These aren't just examples, they are working stock lab solutions. We used to make 5M NaCl all the time and it is soluble

Sigma even sell bottles of the stuff already made up

http://www.sigmaaldrich.com/catalog/sea ... SIAL/S6546
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Postby creepster » Jul 31 2006 12:22 pm

plutoniah wrote:no offense WeridOmen, but you may want to read through John's post. You're right that the solubility of NaCl is 360 g/l at RT, but that means that you can add 360 g NaCl to 1 L water. The final volume will be more than 1 L and thus the concentration will not be 6.15 M.

The actual solublity of NaCl at RT is 4.5 M. That's 264.6 g disolved in a final volume of 1 L. Even at 100 C, the solubility is only 4.8 M.


i dont know the physics of solubility, but i do know that we also have 5M stocks of NaCl in the lab (which would be the needed amount of g in a final volume of 1L)
you can write calculations all you want, maybe we just broke the law of physics that those solution exists even in different parts allover the world

the only thing i'll admit is, that after a couple of years you have precipitated salt in those bottles, but only because water keeps evaporating
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Postby r.rosati » Jul 31 2006 12:57 pm

plutoniah wrote:no offense WeridOmen, but you may want to read through John's post. You're right that the solubility of NaCl is 360 g/l at RT, but that means that you can add 360 g NaCl to 1 L water. The final volume will be more than 1 L and thus the concentration will not be 6.15 M.

The actual solublity of NaCl at RT is 4.5 M. That's 264.6 g disolved in a final volume of 1 L. Even at 100 C, the solubility is only 4.8 M.


No offense taken, but i still stand on my point. Sigma, Ambion and the like all sell 5M NaCl solutions. You can download a data sheet from the Sigma-aldrich site informing that "Maximum solubility of NaCl in water at 25°C is 357 mg/ml. NaCl is unusual in that its solubility does not increase appreciably with temperature, since at 100°C, the solubility is 384 mg/ml." (reference to the Merck index).

On a side note, I'm afraid you're wrong on the way you prepare solutions. Regardless to the fact that many biologists prepare solutions the way you do (because the solutions work anyways), if you want to do things straight, and you're preparing one liter of any lab solution (in water), you should put salts in an appropriate recipient and add water to a final volume of one liter. Adding one liter of water to the salts is simply not correct - it doesn't make a big change in your experiments, but it's wrong. Thus solubility is universally reported as a quantity of solute in a final volume of one liter (you may check any chemistry book), and for this reason, your statement that "you can add 360 g NaCl to 1 L water. The final volume will be more than 1 L and thus the concentration will not be 6.15 M." is not correct.

Sigma, citing the Merck Index, reports NaCl solubility as 357 mg/ml at 25°C. The molecular weight of NaCl is 58.44. A saturated solution of NaCl in water at 25°C is thus approximately 6.11 M.
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Postby kmunson779 » Jul 31 2006 3:41 pm

Our undergrad was making up 5M NaCl before summer break, and couldn't get it to dissolve, even after stirring overnight. I checked her math and she was fairly certain she'd added the correct amount of salt. So, I tried adding just a little bit more water to her solution (she was waiting for the crystals to dissolve before bringing it all the way up to volume) and, like magic, everything dissolved. So 5 M is pushing it, but possible.

Yes, Virginia, there is 5M NaCl...
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Postby John Buckels » Jul 31 2006 5:12 pm

On a slight tangent, I used to use the 5M stock NaCl to gargle with when I had mouth ulcers










that sounded a lot better in my head before I wrote it down :cry:
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Postby kmunson779 » Jul 31 2006 6:59 pm

Not to follow you off on your tangent...

But what the heck? I don't have any experiments running right now!


It seems like gargling with saturated NaCl would be a fast way to get a very dry-feeling mouth...

I find it odd that table salt, if you taste a pinch of it, seems sweet as well as salty -- I haven't figured how that one happens yet.
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