Make 500mL of media using DMEM, 1) 10% FBS, and 1x Pen-Strep. You have stock con
ID: 203780 • Letter: M
Question
Make 500mL of media using DMEM, 1) 10% FBS, and 1x Pen-Strep. You have stock concentrations of 100% FBS and 100x Pen-Strep.
2). I want to do a large scale protein purification. Tris Buffered Saline (TBS) is an essential base for all of the buffers. For now, you need to make 1L of TBS.
The TBS you want to make is made of 200mM NaCl, 20mM Tris (pH 8).
You first need to make 1L stock concentrations of 5M NaCl and 1L of 1M Tris. How will you make those?
Then you need to use those stocks to make your TAE.
Explanation / Answer
Preparation of 500mL of media using DMEM contain 10% FBS, and 1x Pen-Strep. You have stock concentrations of 100% FBS and 100x Pen-Strep.
1.FBS
FBS stock concentration = 100%
Final FBS concentration in media = 10%
Final volume = 500ml
M1V1 (Concentrated) = M2V2 (Diluted)
100% * xml = 10%*500ml
xml = 10%*500ml/100%
= 50ml
We have to take 50ml from 100% stock to get 10% for 500ml media
100x Pen-Strep
Stock Pen-Strep concentration = 100X
Required Pen-Strep concentration = 1X
M1V1 (Concentrated) = M2V2 (Diluted)
100X * xml = 1X*500ml
xml = 1X*500ml/100X
= 5ml
We have to take 5ml from 100 X Pen-Strep stocks to get 1x for 500ml media.
For to 500ml media:
S.No.
Component
Final concentration
Amount needed for 30ul reaction
1
FBS 100% stock
10%
50ml
2
100X Pen-Strep
1X
5ml
4
Mol grade H2O
-
445ml
Total volume 500ml
Molarity: it is a measure of the concentration of solute in a solution. It is defined as a number of moles of solute present in 1 liter of solution.
Molarity = no of moles/volume of solution
M = n/V (in liters)
If n = weight (wt) /Molecular weight (M.Wt)
Then
M = wt/ M.Wt *1/ V (in liters)
Or
M = wt/ M.Wt *1000 / V (in milliliters)
Stock A) preparation of one liter of 5M NaCl (FW of NaCl is 58.4g/mol)
one liter of 5M NaCl stock solution preparation:
Weight of solute = (x)? gm
Molecular weight of glucose =58.4g/mol
Volume of solution =1000ml or 1 liter
Molarity (M) = wt/ M.Wt *1000 / V (in milliliters)
5M= x gm/ 58.4*1000 / 100 (in milliliters)
x gm=5M*58.4*1000/1000
=292gm
Take 292 gm of NaCl and dissolve in the 1000ml of double distilled water then you will get 1000ml of 5M NaCl solution.
Stock B) preparation of one liter of 1M tris (FW of trise is 121.14g/mol)
one liter of 1M tris stock solution preparation:
Weight of solute = (x)? gm
Molecular weight of glucose =121.14g/mol
Volume of solution =1000ml or 1 liter
Molarity (M) = wt/ M.Wt *1000 / V (in milliliters)
1M= x gm/ 121.14*1000 / 100 (in milliliters)
x gm=1M*121.14*1000/1000
=121.14gm
Take 121.14 gm of tris and dissolve in the 1000ml of double distilled water then you will get 1000ml of 1M tris solution.
1 liter TBS with the 200mM NaCl, 20mM Tris (pH 8) final concentrations
NaCl, 200mM
NaCl 5M stock concentration = 5M/5000mM
Final Nacl concentration = 200mM
Final volume = 1000ml
M1V1 (Concentrated) = M2V2 (Diluted)
5000mM * xml = 200mM*1000ml
xml = 200mM*1000ml/5000mM
= 40ml
We have to take 40ml from NaCl 5M stock to get 200mM for 1000ml solution
tris, 20mM
tris 1M stock concentration = 1M/1000mM
Final tris concentration = 20mM
Final volume = 1000ml
M1V1 (Concentrated) = M2V2 (Diluted)
1000mM * xml = 20mM*1000ml
xml = 20mM*1000ml/1000mM
= 20ml
We have to take 20ml from tris 1M stock to get 20mM for 1000ml solution
For to 1000ml TBE solution:
S.No.
Component
Final concentration
Amount needed for 30ul reaction
1
NaCl 5M stock
200mM
40ml
2
Tris 1M stock
20mM
20ml
4
Mol grade H2O
-
940ml
Total volume 500ml
S.No.
Component
Final concentration
Amount needed for 30ul reaction
1
FBS 100% stock
10%
50ml
2
100X Pen-Strep
1X
5ml
4
Mol grade H2O
-
445ml
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