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 plasmid vector to be used in total will be 0.1 µg. The final reaction volume will be 20 µL. The final concentration of the 10X ligase buffer should be 1X. Using this information, complete the following chart regarding the volumes of each component used to prepare your samples. (Hint: For the plasmid and insert, determine the mass of DNA that needs to be in each tube using the information given, then use the equation C=mass/V to solve for the volume.) Ratio Volume of 0.05 µg/µL Plasmid Vector (µL) Volume of 0.2 µg/µL Insert DNA (µL) Volume of 10X Ligase Buffer (µL) Volume of Water (µL) Total Volume 3:1 20 µL 6:1 20 µL.

Expert Solution

Given that we need to use 0.1ug of Plasmid vector. The concentration of DNA in the stock solution is 0.05ug/ul.

So the volume of plasmid vector = 0.1/0.05 - 2ul.

Given that concentration of Insert DNA (0.2 µg/µL).

Volume for 3:1 ratio - 3:1 ratio means the amount of insert is 3 times of plasmid vector.

Amount of plasmid vector = 0.1ug.

So the amount of insert = 3*0.1 = 0.3ug.

The volume of insert = Amount of insert/concentration of insert = 0.3/0.2 = 1.5ul.

Volume for 6:1 ratio - 6:1 ratio means the amount of insert is 6 times of plasmid vector.

Amount of plasmid vector = 0.1ug.

So the amount of insert = 6*0.1 = 0.6ug.

The volume of insert = Amount of insert/concentration of insert = 0.6/0.2 = 3 ul.

	3: 1 ratio	6:1 ratio
Insert DNA (µL)	1.5	3
Volume of 10X Ligase Buffer (µL)	2	2
Volume of Water (µL)	13.5	12
Volume of 0.05 µg/µL Plasmid Vector (µL)	2	2
T4 DNA ligase (µL)	1	1
Total volume (µL)	20	20

Please write to me if you need more help.

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 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

Part 2: Complete Ligation Reaction Calculations (10 marks) You are setting up a ligation reaction with...

Part 2: Complete Ligation Reaction Calculations You are setting up a ligation reaction with the following components: Plasmid Vector (0.02 µg/µL) Insert DNA (0.03 µg/µL) 10X T4 Ligase Buffer T4 DNA Ligase Enzyme Water You plan to set up one experiment with a _______ ratio of insert to plasmid. The amount of plasmid vector to be used in total will be _____ µg. The final reaction volume will be 50 µL. You will add 2.5 µL of T4 Ligase once...

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...

Discuss the following research techniques 1. DNA ligase 2. Plasmid vector

What are the components of agarose gel electrophoresis set up?

3) How can you use PCR to clone a 2000bp gene into a plasmid vector?

You decide to clone a gene using a plasmid vector featuring ampicillin resistance and the lacZ...

You decide to clone a gene using a plasmid vector featuring ampicillin resistance and the lacZ gene. However, when you try to grow the E. coli onto a plate containing X-gal and ampicillin, you see only blue colonies. Explain what most likely went wrong during the cloning process.

2. Set up both the vector of state probabilities and the matrix of transition probabilities given...

2. Set up both the vector of state probabilities and the matrix of transition probabilities given the following information: Store 1 currently has 40% of the market; store 2 currently has 60% of the market. In each period, store 1 customers have an 80% chance of returning; 20% of switching to store 2. In each period, store 2 customers have a 90% chance of returning; 10% of switching to store 1. a. Find the percentage of market for each store...

Consider the following experiment: a double-slit set-up for firing electrons one at a time. Let's now...

Consider the following experiment: a double-slit set-up for firing electrons one at a time. Let's now add a second electron (orange), which is fired parallel to the first one, but in the opposite direction, and so as to pass closer to one slit than the other, and, importantly, above the plane in which interference occurs: The firing of the two electrons is timed so that the orange electron is "closest" to the purple electron somewhere around point P, i.e. after...

Question