11. A buffer used to denature proteins include the following components: a. 25 m
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Question
11. A buffer used to denature proteins include the following components: a. 25 mM Tris-Hcl, pH 8.2 b. 8 M Urea c. 200 mM Glycine d. 10 mM beta-mercaptoethanol e. 250 mM NaC In the laboratory you have a 1 M solution of Tris buffer at pH 7.0, a bottle of beta- mercaptoethanol at 14.3 M and solid urea, glycine and NaCl. Calculate how you would prepare this buffer. Describe the making of the buffer with details including rinsing glasses, how you would measure each item, volumes of water added to the beakers and so on...Explanation / Answer
Given,
1 M solution of Tris buffer at pH 7.0,
a bottle of beta- meractoeethanol at 14.3 M
solid urea
glysine
NaCl.
Required to prepare,
a. 25 mM Tris- Hcl, pH 8.2
b. 8M Urea
c. 200 MM glycine
d. 10mM beta- meractoeethanol
e. 250 mM NaCl
Lets prepare a 1 Liter solution of it.
1 M solution of Tris buffer at pH 7.0 to 25 mM Tris- Hcl, pH 8.2. pH can be adjusted before adding itself by using NAOH palletes.
We know that, N1V1=N2V2
we have, 1M.X=25mM.1000ml
So the volume to be used from stock X=25 * 1000/1000 (1M is 1000 mM)
Therefore, 25 ml of stock to be diluted upto 1000 ml to get 25 mM of tris is obtained.
Now beta- meractoethanol at 14.3 M to 10mM beta- meractoeethanol
=> 14.3*V1=10mM*1000
V1=10*1000/14300 = 0.6993 mL
So, 0.6993 mL of stock to be make upto 1000 mL.
8M Urea for 1L preparation:
MW of urea = 60
So for prepartion for 8M 8*60 = 480 g of urea is required.
For 200 mM glycine in 1L, MW of glycine 75
so we need 75 g in 1000 ml for 1M (1000mM)
Therefore, 75/5 = 15 g in 1000 for 200 mM i srequired.
For 250mM NaCl:
The MW of NaCl =58.5 => 58.5 / 4 = 14.63 g of NaCl in 1000 ml to get 250 mM is required.
We get 25 ml of stock of tris to be diluted upto 1000 ml to get 25 mM of tris, 0.6993 ML OF STOCK beta- meractoeethanol to be make upto 1000 ml, need 15 g of glycien in 1000 for 200mM, 14.63 g of NaCl in 1000 ml to get 250mM, 480 g of urea in 1000 ml. Take all the required things into a glass jar and 1000 ml of volume is made.
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