Your lab instructor has given you a protocol to perform a molecular cloning expe

Question

Your lab instructor has given you a protocol to perform a molecular cloning experiment. In a previous experiment, you used polymerase chain reaction (PCR) to amplify a sequence that you believe to regulate expression of a gene you are studying. You will now take this purified PCR product (double stranded DNA) and ligate it into a plasmid that contains a luciferase reporter gene. If your DNA sequence is a promoter sequence, then its presence will allow for expression of the luciferase reporter gene when transfected into a human cell line. The goal of this experiment is to ligate the PCR product into the luciferase reporter plasmid, transform it into E. coli, and then column purify plasmid that can be transfected into human cells in culture.

The first step of this experiment will be to determine the concentration of DNA in your luciferase reporter plasmid tube and in your purified PCR product tube. You use a spectrophotometer and determine the concentration of the luciferase reporter plasmid DNA to be 1.56 mg/ml and the concentration of the PCR product to be 200 mg/ml. Next, you must prepare your ligation reaction. The ligation reaction has a final volume of 20ml and the manufacturer recommends the following final reaction composition: 1x ligation reaction buffer, 100ng total plasmid DNA, 300ng total PCR product, 10 units total T4 DNA ligase, and the remaining volume should be molecular grade water. The ligation kit came with tubes labeled: “10x ligation reaction buffer”, “molecular grade water”, and “100,000 units/ml T4 DNA ligase”. Using this information, you will need to set up the ligation reaction for this part of the experiment.

Question 1

A: What is the final ligation reaction volume?

B: Fill in this table with the relevant information.

Ligation Reaction

Initial Concentration (Ci)

Final Concentration (Cf)

Ligation Reaction Buffer

Plasmid DNA

PCR Product

T4 DNA Ligase

Question 2 (3pts)

Convert the units for each variable (A-C below) from the initial concentration units to the units listed below.

A: Plasmid DNA (convert to ng/ml):

B: PCR Product (convert to ng/ml):

C: T4 DNA Ligase (convert to units/ml):

Question 3 (3pts)

A: If you set up the exact ligation reaction described in the instructions, what volume (ml; microliters) of ligation reaction buffer would you add to the tube?

B: If you set up the exact ligation reaction described in the instructions, what volume (ml; microliters) of T4 DNA ligase would you add to the tube?

Question 4 (4pts)

A: If you set up the exact ligation reaction described in the instructions, what volume (ml; microliters) of luciferase reporter plasmid DNA would you add to the tube?

B: If you set up the exact ligation reaction described in the instructions, what volume (ml; microliters) of PCR product would you add to the tube?

Ligation Reaction

Initial Concentration (Ci)

Final Concentration (Cf)

Ligation Reaction Buffer

Plasmid DNA

PCR Product

T4 DNA Ligase

Explanation / Answer

A: What is the final ligation reaction volume?

Final ligation reaction volume is 20ml

B: Fill in this table with the relevant information.

Ligation Reaction

Initial Concentration (Ci)

Final Concentration (Cf)

Ligation Reaction Buffer

Plasmid DNA

PCR Product

T4 DNA Ligase

2- 1mg = 1000microgram

1 microgram = 1000 nanogram.

So 1 mg = 1000000ng or 10^6ng

1.56 mg/ml and the concentration of the PCR product to be 200 mg/ml.

A: Plasmid DNA (convert to ng/ml): given that the concentration of plasmid DNA 1.56mg/ml so its concentration will be 1.56*10^6ng/ml

B: PCR Product (convert to ng/ml): Concentration of PCR product is 200mg/ml. Its concentration will be 200 *10^6ng/ml

C: T4 DNA Ligase (convert to units/ml): Concentration of T4 DNA ligase is 100000 unit/ml.

3-

A: If you set up the exact ligation reaction described in the instructions, what volume (ml; microliters) of ligation reaction buffer would you add to the tube?

Since the ligation buffer is 10X so we need to use 2ml of 10X ligation buffer in 20ml of reaction volume

B: If you set up the exact ligation reaction described in the instructions, what volume (ml; microliters) of T4 DNA ligase would you add to the tube?

Stock T4 DNA ligase is 100000unit/ml and we need to use 10unit for the reaction. So we need to use 0.1microlitres.

1ml = 1000 microlitres

Question 4 (4pts)

A: If you set up the exact ligation reaction described in the instructions, what volume (ml; microliters) of luciferase reporter plasmid DNA would you add to the tube?

Concentration of plasmid is 1.56*10^6ng/ml or 1.56*10^3ng/microlitres

We need to use 100ng of a plasmid. SO we will add 0.06microlitres of plasmid DNA

B: If you set up the exact ligation reaction described in the instructions, what volume (ml; microliters) of PCR product would you add to the tube?

Concentration of plasmid is 200*10^6ng/ml or 200*10^3ng/microlitres

We need to use 300ng of a PCR product. SO we will add microlitres of plasmid DNA 0.0015 microliter of plasmid

Ligation Reaction

Initial Concentration (Ci)

Final Concentration (Cf)

Ligation Reaction Buffer

10X 1X

Plasmid DNA

1.56mg/ml 100ng/20ml or 5ng/ml

PCR Product

200mg/ml 300ng/20ml or 15ng/ml

T4 DNA Ligase

100000unit/ml 10unit/20ml or 0.5unit/ml

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