Question

Determine the amounts of the solutes necessarry to prepare the indicated solutions.

A) 4L Dialysis Buffer (25mM HEPES, 0.01% (v/v) Triton X-100, 300mM NaCl, 2.5M Urea)

B) 250mL 25x MES running buffer (1x= 50mM MES, 50mM Tris, 0.1% (w/v) SDS, 1mM EDTA)

C) 1:15,000 SYBR Green (25mg/mL0 in 80mL 0.8% agarose

D) What is the final micromolar concentration of SYBR Green in the agarose solution in Part C?

Formula Masses

Tris: 121.14

NaCl: 58.44

HEPES: 238.3

Urea: 60.06

MES: 195.24

EDTA:292.24

SYBR Green: 509.73

Answer 1

Answers :

A) According to question, 4L Dialysis Buffer contains 25mM HEPES, 0.01% (v/v) Triton X-100, 300mM NaCl and 2.5M Urea.

25mM HEPES indicates the presence of 25mM HEPES in 1L solution. Here the amount of solution is 4L. Hence amount of HEPES is (25mM*4) = 100 mM = 100 mmol. Now, the formula mass of HEPES is 238.3 g/mol = 238.3 mg/mmol. Hence, 100 mmol contains (100*238.3) mg = 23830 mg = 23.83 g

0.01% (v/v) Triton X-100 indicates the presence of 0.01%*4L = 0.0004 L = 0.4 ml Triton X-100. Density of Triton X-100 is 1.07 g/ml (not given in question, taken from textbook). Hence the mass of 0.4 ml Triton X-100 is 1.07*0.4 g = 0.428 g

300mM NaCl indicates the presence of (300mM*4) = 1200 mM = 1.2 moles of NaCl in 4L solution. The calculation above is similar like Triton X-100. Now the molar mass of NaCl is 58.44 g/mol as given in the question. Hence mass NaCl present in 4L solution is 1.2 moles * 58.44 g/mol = 70.128 g

2.5M Urea indicates the presence of (2.5M*4) = 10 M = 10 moles of Urea in 4L solution. The calculation above is similar like Triton X-100. Mass of Urea present in 4L solution is 10 moles * 60.06 g/mol = 600.6 g

Hence 4L Dialysis Buffer contains 23.83 g HEPES, 0.428 g Triton X-100, 70.128 g NaCl and 600.6 g Urea.

B) In 250mL 25x MES running buffer (1x= 50mM MES, 50mM Tris, 0.1% (w/v) SDS, 1mM EDTA), we will calculate the amounts in 1X first, then we will multiply with 25 to get the amount in 25X MES Buffer.

Amount of Buffer = 250 ml = 0.25L

50mM MES indicates the presence of 50 mmol MES in 1L solution. In 0.25L the amount will be 50*0.25 mmol = 12.5 mmol. Formula mass of MES is 195.24 g/mol = 195.24 mg/mmol. Mass of MES is (12.5*195.24) mg = 2440.5 mg = 2.4405 g. Now, this calculation is for 1X Buffer. For 25X Buffer, the mass of MES will be 2.4405 g*25 = 61.0125 g.

50mM Tris indicates the presence of 50 mmol Tris in 1L solution. In 0.25L the amount will be 50*0.25 mmol = 12.5 mmol. Formula mass of MES is 121.14 g/mol = 121.14 mg/mmol. Mass of MES is (12.5*121.14) mg = 1514.25 mg = 1.51425 g. Now, this calculation is for 1X Buffer. For 25X Buffer, the mass of MES will be 1.51425 g*25 = 34.85625 g.

0.1% (w/v) SDS indicates the presence of 0.1%*0.25 g SDS = 0.00025 g SDS. Now, this calculation is for 1X Buffer. For 25X Buffer, the mass of SDS will be 0.00025*25 g = 0.00625 g = 6.25 mg.

1mM EDTA indicates the presence of 1mmol/L*0.25L = 0.25 mmol EDTA. Formula mass of EDTA is 292.24 g/mol = 292.24 mg/mmol. Mass of EDTA is (0.25*292.24) mg = 73.06 mg. Now, this calculation is for 1X Buffer. For 25X Buffer, the mass of EDTA will be 73.06 mg*25 = 34.85625 g.

250mL 25x MES running buffer contains 61.0125 g MES, 34.85625 g TRIS, 6.25 mg SDS, 34.85625 g EDTA.

C) In 1:15,000 SYBR Green solution 1:15,000 indicates dilution. It says in total 15000 parts of solution, only 1 part of SYBR Green is present. Here we will first calculate the amount of SYBR Green and Agarose in 80 ml solution as given in the question, then we will divide the amount by 15000 to get actual mass of both SYBR Green and Agarose.

Concentration of SYBR Green = 25 mg/ml. Hence 80 ml will constitute 25*80 mg = 2000 mg. This is for 1 part. Now 1/15000 parts the amount will be 2000/15000 mg = 0.133 mg.

Concentration of Agarose is 0.8%. 100 ml solution contains 0.8 g Agarose. 80 ml solution will contain 80*0.8 g = 64 g Agarose. This is for 1 part. Now 1/15000 parts the amount will be 64/15000 g = 0.004266 g = 4.266 mg.

Hence 1:15000 SYBR Green solution contains 0.133 mg SYBR green and 4.266 mg Agarose.

D) Molar mass of SYBR green = 509.73 g/mol . Mole amount of 2000 mg or 2 g SYBR green = 2/509.73 mol = 0.00392 mol. This is present in 80 ml solution. Hence concentration will be 0.00392/80 moles per ml = 0.000049 moles per ml = 0.049 moles/L.

Now in 1:15000 solution, the concentration will be 0.049/15000 moles/L = 3.266 * 10^-6 M = 3.266 µM (micromolar).

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