Suppose that another transformation experiment was conducted. In this experiment

Question

Suppose that another transformation experiment was conducted. In this experiment, 5 µl of a plasmid suspension with a concentration of 0.61 ng/µl was added to 45 µl of competent cells. The mixture was incubated on ice, subjected to temperature shock and then added to 950 µl of Luria broth. The solution was then incubated for 1 hr.

An aliquot of 85 µl was spread on a plate containing ampicillin and X-gal. This plate was labeled "undiluted.”

A tube was prepared by making 70 µl of a 1/5 dilution of the transformed cells and all 70 µl of the diluted solution was spread on a second plate with ampicillin and X-gal. This plate was labeled "diluted.”

The plates were scored after incubation at 37oC for 16 hrs. Results are given below. Assume that the plasmid suspension used in this experiment was obtained from a ligation reaction in which attempts were made to insert a hippopotamus gene into the MCS of a pUC19 vector (2686 bp). (Remember that pUC19 contains an ampicillin resistance gene and the LacZ gene. The MCS is in the LacZ gene.) Round final answers to four decimal places. Do not use scientific notation.

In making the 70 µl of diluted culture, how many µl of culture was added to the tube?

Answer:

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In making the 70 µl of diluted culture, how many µl of broth was added to the tube?

Answer:

Question 23

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What is the cloning efficiency (as a percentage) for the diluted plate?

Answer:

Question 24

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How much DNA (in nanograms) was used to transform the cells on the diluted plate?

Answer:

Question 25

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What is the transformation efficiency (ug-1) for the diluted plate? (Do not use scientific notation).

Answer:

What is the transformation efficiency (ug-1) for the diluted plate? (Do not use scientific notation).

Answer:

Question 26

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What is the cloning efficiency (as a percentage) for the undiluted plate?

Answer:

Question 27

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How much DNA (in nanograms) was used to transform the cells on the undiluted plate?

Answer:

Question 28

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What is the transformation efficiency (ug-1) for the undiluted plate? (Do not use scientific notation).

Answer:

Explanation / Answer

Question: In making the 70 µl of diluted culture, how many µl of culture was added to the tube?

Answer: It is given that 85 µl is undiluted.

And the total volume of undiluted culture = 1000µl

And the dilution is 1/5 times

Which means that the undiluted culture is 5X of diluted culture

Applying formula M1 V1=M2 V2

5x XV1 = x X 70

V1=70/5

V1 = 14 µl

So to make 70 In making the 70 µl of diluted culture, how many µl of broth was added to the tube of diluted culture The volume of culture to be take will be 14µl.

Question: In making the 70 µl of diluted culture, how many µl of broth was added to the tube?

To make 70 µl of diluted sample the 14 µl main culture is to be make up with broth or which will be diluted to make up volume upto 70 In making the 70 µl of diluted culture, how many µl of broth was added to the tube

So, 70 µl -14 µl =56 In making the 70 µl of diluted culture, how many µl of broth was added to the tube water will be added to 14 µl culture.

In making the 70 µl of diluted culture, 56 many µl of broth was added to the tube

2. Question

What is the cloning efficiency (as a percentage) for the diluted plate?

Answer:

Cloning efficiency = (no. of transformed clones or white colonies/total no. of colonies)*100

= (19/19+92)*100

= 19/111*100

Cloning efficiency = 17.11%

Question: How much DNA (in nanograms) was used to transform the cells on the diluted plate?

Answer: Given that,

5 µl of 0.61 ng/µl is the DNA used in undiluted reaction of 1ml

Therefore total DNA in 1000 µl of total reaction undiluted = 5 X 0.61 = 3.05 ng

Therefore for 1V of volume the quantity of DNA = 3.05/1000*14

=0.0427 ng.

So for 70 µl of diluted sample

We have taken 14 µl of the undiluted sample which contain 0.0427 ng of DNA.

So, to transform the cells in diluted samples of 70 µl the amount of DNA = 0.0427 ng.

Question: What is the transformation efficiency (ug-1) for the diluted plate? (Do not use scientific notation).

Answer: No. of white colonies/ amoumt of DNA in µg

=19/0.0427*10-3

=44496

=4.4X106.

Q,Q 26,27 and 28THe procedure will be same for the rest undiluted samples A above 4 question taking the values of undiluted samples.

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