Question

In: Biology

You want a 50 µL reaction volume containing 1X reaction buffer, 1 µg DNA, and 10...

You want a 50 µL reaction volume containing 1X reaction buffer, 1 µg DNA, and 10 Units of enzyme.You have a plasmid with a concentration of 250 ng/µL, 10X reaction buffer, and your enzyme concentration is 20,000 units/mL.

How many µL of water, reaction buffer, DNA and enzyme would you combine to set up that reaction? Please show work

Expert Solution

Total volume of reaction= 50 microliters

a) Given is 10X reaction buffer

Hence, to obtain 1X concentration, 10X buffer should be diluted 1: 10

C1V1=C2V2

10X X V1= 1X X 50 microliters

V1= 1 X 50/10= 5 microliters

5 microliters of 10X reaction buffer is added

b) Required DNA concentration is 1 microgram DNA/50 microliters

Given is 250 ng/microliters of plasmid DNA

1ng = 0.001 microgram

250 ng= 250 X 0.001= 0.25 microgram

Plasmid DNA= 0.25 microgram/ microliters

Amount of plasmid DNA added= 0.25 x 4= 1 microgram

Hence, 4 ml of 250 ng/ microliters of plasmid DNA is added to give a final concentration of 1 microgram of DNA in total volume of 50 microliters.

c) Given stock enzyme concentration is 20,000 units/ml

Required enzyme concentration is 10 units/50 microliters

Enzyme concentration in stock= 20000 units/ ml= 20000 units/ 1000 microliters = 20 units/ microliters

Hence, 0.5 microliters (10 units/ microliter) will be added to the total 50 microliters reaction volume to obtain 10 units/ microliters of enzyme

d) Distilled water= 50 microliters – (volume of enzyme + DNA + reaction buffer)

Distilled water= 50 –(5+4+0.5)= 50-9.5= 40.5 microliters

Final reaction of 50 ml:

5 microliters of 10X reaction buffer

4 microliters of 250 ng/ microliter plasmid DNA

0.5 microliters of 20000 units/ml enzyme

40.5 microliters of distilled water

gladiator answered 2 years ago

7.You want to set up a PCR reaction. The protocol suggests a 50 µL reaction volume...

7.You want to set up a PCR reaction. The protocol suggests a 50 µL reaction volume containing 1X buffer, 200 µM dNTPs, 0.5 µM each primer (forward and reverse), 250 ng template DNA, and 1 U enzyme. You have the following reagents: 5 X buffer, 10 mM dNTPs, 10 µM each primer (forward and reverse), 125 ng/µL template DNA, and 2 U/µL enzyme. How many µL of water, reaction buffer, dNTPs, each primer, template DNA and enzyme would you combine...

You now set up a ligation reaction with the following components: • Plasmid Vector (0.05 µg/µL)...

You now set up a ligation reaction with the following components: • Plasmid Vector (0.05 µg/µL) • Insert DNA (0.2 µg/µL) • 10X Ligase Buffer • Water Because you are unsure what the best ratio of insert to plasmid is, as this is a new experiment for you, you plan to set up two experiments, one with a 3:1 ratio of insert to plasmid, and one with a 6:1 ratio of insert to plasmid. In both cases the amount of...

you want to make 100ml of 0.20M acetic acid buffer with pH=50. you are given a...

you want to make 100ml of 0.20M acetic acid buffer with pH=50. you are given a stock of 1.0M acrtic acid and a bottle of solid sodium acetate sale (MW=82g/mol). what is the ratio of [A-]/[HA] when your buffer pH is 5.0? determine the concentration of weak acid and conjugate base in the buffer. how many mL of 1.0 acetic acid will you need? how many grams of sodium acetate will you need? if you now have add 1mL of...

1. Consider a reaction mixture containing 100.0 mL of 0.112 M borate buffer at pH =...

1. Consider a reaction mixture containing 100.0 mL of 0.112 M borate buffer at pH = pKa = 9.24. At pH = pKa, we know that [H3BO3] = [H2BO3−] = 0.0560 M. Suppose that a chemical reaction whose pH we wish to control will be generating acid. To avoid changing the pH very much we do not want to generate more acid than would use up half of the [H2BO3−]. a)How many moles of acid could be generated without using...

if you want to prepare a buffer at pH = 5.2 which buffer would you choose?...

if you want to prepare a buffer at pH = 5.2 which buffer would you choose? a) MES --> Ka = 7.9 x 10^-7 b) CHES --> Ka = 5.0 x 10^-7 c) Tris --> Ka = 6.3 x 10^-9 d) acetic aid --> Ka = 1.8 x 10^-5

10) You want to create a buffer using phosphoric acid and Sodium Dihydrogen Phosphate. what is...

10) You want to create a buffer using phosphoric acid and Sodium Dihydrogen Phosphate. what is the best pH at which to make the buffer? using that pH and a concentration of 1M. Determine the molar concentrations of each component (acid and base) needed to make the buffer. If you need 100mL of the buffer, how many mL of 2.5 M phosphoric acid will you need? How many grams of the Sodium Dihydrogen Phosphate do you need? 11) using the...

A mixture of hydrocarbon gaseous, containing on a volume basis: 80% methane, 10% ethane and 10%...

A mixture of hydrocarbon gaseous, containing on a volume basis: 80% methane, 10% ethane and 10% n-propane are burned in a boiler where saturated steam are produced at 1 atm. The gas mixtures enters the boiler at a rate of 100 m 3 /hr (STP) and 25oC and the water enters the boiler at 25oC. The percentage conversion of methane is 90%; of the methane burned, 75% reacts to form carbon dioxide and the balance reacts to form carbon monoxide;...

A student dilutes 50 mL of a phosphoric acid-dihydrogen phosphate buffer solution to a final volume...

A student dilutes 50 mL of a phosphoric acid-dihydrogen phosphate buffer solution to a final volume of 100 ml. Will the pH of the buffer change noticeably? (Use Henderson -Hasselbeck conceptually ) For the same dilution above will the buffer capacity change ? Calculations: A 50.0 mL sample of a 0.100 M phosphoric acid, H3PO4 is titrated with 0.100 M NaOH. Identify the major chemical species present and calculate the pH at these four points (first consider the titration curve)...

Question 1: You set up a 200 µL PCR to amplify a 1-kb fragment by adding...

Question 1: You set up a 200 µL PCR to amplify a 1-kb fragment by adding 20 µL of a 500 nM primer that is 20-bp in length. How many times would you expect to find the exact 20-bp sequence in the human genome (using a genome size of 3.0 E+9 bp) at random? How many moles of the primer have you added? How many molecules of that primer have you added? Describe whether this amount of primer is a...

1.) Medication ordered: Prepare 500 ml of a 10% solution -available: 50% solution -What volume of...

1.) Medication ordered: Prepare 500 ml of a 10% solution -available: 50% solution -What volume of the 50% solution is needed? 2.) Medication ordered: Prepare 250ml of a 15% solution Available: 70% solution What volume of the 70% solution is needed? 3.) Medication ordered: Prepare 600 ml of a 25% solution Available: 75% solution is needed? a. What volume of the 75% solution is needed? 4.) Medication ordered: Prepare 750 ml of a 20% solution Available: 80% solution What volume...