A)Describe how you would prepare a 1X TBE electrophoresis buffer using the 10X T
ID: 50995 • Letter: A
Question
A)Describe how you would prepare a 1X TBE electrophoresis buffer using the 10X TBE as a stock solution assuming you need a final volume of 800 ml.
B) Assume that a 0.5% (w/v) stock solution is available in the lab that you will use to make up three different 5 ml working solutions. The concentration of each of the working solutions is as follows: 0.25%, 0.05%, and 0.001%. Calculate the volume of stock solution and water that will be used for each dilution. Indicate which micropipette you would use to make each working solution.
C) Assume that you need to mix a 1 mL stock solution of 10X restriction buffer with the following final concentration: 600 mM Tris, 100 mM MgCl, 70 mM DTT. Further assume that you have the following solutions available to you: 2 M Tris, 1M MgCl, and 1.5 M DTT. Describe the preparation of the 10X restriction buffer showing all calculations. Describe how you would dilute this 10X restriction buffer to 2X with a volume of 50 microliters. Remember these volumes are much too small for volumetric flasks.
Explanation / Answer
A)Describe how you would prepare a 1X TBE electrophoresis buffer using the 10X TBE as a stock solution assuming you need a final volume of 800 ml.?
Answer: when diluting solutions use the following formula.
C1V1=C2V2 (OR) M1V1=M2V2(OR)N1V1=N2V2
C1=concentraion of initial stock soltion.
V1=Volume to be taken from the Stock solution
C2= Concentrion required of working soltion
V2=Volume required of working soltion.
M1=molarity of the STOCK solution
M2=molarilty of Working solution
N1=normality of the STOCK solution
N2=normality of Working solution
(Note:stock solution- already prepared and high concentrated solution,working solution-to be prepared and less concentrated solution)
C1V1=C2V2 is applied for the above dilution
If substitute values in the above formula
10X *?=1X*800ml
V1=80ml
take 80ml of stock solution 10X TBE buffer and make up the volume with 720ml of Distilled water to get a final concentrion of 1X TBE buffer of 800ml .
(80ml of stock solution 10X TBE buffer + 720ml of Distilled water)
B) Assume that a 0.5% (w/v) stock solution is available in the lab that you will use to make up three different 5 ml working solutions. The concentration of each of the working solutions is as follows: 0.25%, 0.05%, and 0.001%. Calculate the volume of stock solution and water that will be used for each dilution. Indicate which micropipette you would use to make each working solution.
Here also we use the same fomula C1V1=C2V2
Calculation for 0.25% of 5ml Solution.
0.5*V1=0.25*5ml
2.5ml
Take 2.5ml of 0.5% stock solution and make to 5ml with distilled water.
(2.5 ml of 0.5% stock solution+2.5ml distilled water)
Calculation for 0.05% of 5ml Solution.
0.5*V1=0.05*5ml
0.5ml
Take 0.5ml of 0.5% stock solution and make to 5ml with distilled water.
(0.5 ml of 0.5% stock solution+4.5ml distilled water)
Calculation for 0.05% of 5ml Solution.
0.5*V1=0.001*5ml
0.01ml
Take 0.01ml (10 microlitres)of 0.5% stock solution and make to 5ml with distilled water.
(0.01ml of 0.5% stock solution+4.99 ml distilled water)
C) Assume that you need to mix a 1 mL stock solution of 10X restriction buffer with the following final concentration: 600 mM Tris, 100 mM MgCl, 70 mM DTT. Further assume that you have the following solutions available to you: 2 M Tris, 1M MgCl, and 1.5 M DTT. Describe the preparation of the 10X restriction buffer showing all calculations. Describe how you would dilute this 10X restriction buffer to 2X with a volume of 50 microliters. Remember these volumes are much too small for volumetric flasks.
M1V1=M2V2 formula is applied here as the stock concentrations are mentioned in molarity.
to prepare 10X from 2 M Tris, 1M MgCl, and 1.5 M DTT.???
M1V1=M2V2
Preparation of 600 mM Tris?
2M*V1=600 mM Tris (working soltion concentrion)*1ml(required volume)
2M*V1=600*10-3 (convertion to molarity)*1ml
V1=0.3ml
Take 0.3 ml of 2M stock solution Tris and make to 1ml with distilled water.
Preparation of 100mM?
1M*V1=100 mM Mgcl (working solution concentrion)*1ml(required volume)
1M*V1=100*10-3 (convertion to molarity)*1ml
0.1ml
Take 0.1 ml of 1M stock solution Mgcl and make to 1ml with distilled water.
Preparation of 1.5 M DTT?
1.5M*V1=70 mM DTT (working solution concentrion)*1ml(required volume)
1.5M*V1=70*10-3 (convertion to molarity)*1ml
0.046
Take 0.046 of 1.5 M stock solution DTT and make to 1ml with distilled water.
WE HAVE TO PREPARE 10X WITH 600 mM Tris, 100 mM MgCl, 70 mM DTT SO,
As we got answer for different diluton now we wil make 10X 1ml buffer
Take 0.3 ml of 600 mM Tris+0.1 ml MgCl+0.046 ml of DTT from their respectve stock slutions(2 M Tris, 1M MgCl, and 1.5 M DTT) and make up to 1ml with Distilled water.
(0.3 ml of 600 mM Tris+0.1 ml MgCl+0.046 ml of DTT+0.554 distlled water)
how to prepare 2x buffer from 10x buffer?
here we use C1V1=C2V2
10X*V1=2X*50microliters
V1=10microliters
Take 10microliters of 10X buffer and add 40 microliters of Distilled water.
Note:As these volumes are much too small for volumetric flasks we Can use pipette for dilutions.