Question

QUESTION 2 OF 10 Please read Chapter 7 of the Lab Manual and the relevant Power Point lecture, and answer the following question. Deadline for submission: April 2, 2020. Cloning 2 Below is the restriction map of a 10 kb piece of DNA. Also shown below is a cloning vector which has two unique restriction enzyme recognition sites, one for EcoRI (E) and one for HindIII (H). The location of the kanamycin (kan) and ampicillin (amp) resistance genes is also shown. Kanamycin and ampicillin are antibiotics that are commonly used to select transformed E. coli cells (consult the Lab Manual for more information). Note that the HindIII site is located within the amp gene. The Ori region is not shown for simplicity. BP HE SB N et 119111 DNA of interest amp" kan' Vector In order to clone the 1.9 kb fragment of the DNA of interest into the vector, you performed the following experiments: 1. digested the DNA of interest with EcoRI and HindIII to retrieve the 1.9 kb fragment 2. digested the vector with the same enzymes 3. mixed the digested DNA and the digested vector together and ligated, thus you created a recombinant vector 4. transformed kanamycin-sensitive E. coli competent cells with the recombinant vector and grew in SOB medium 5. plated the transformants on an agar plate containing LB medium supplemented with kanamycin and Xgal (LB + kan + Xgal), and incubated the plate at 37°C in order to select kanamycin-resistant white colonies Question: If you plate the selected kanamycin-resistant transformants (step 5 above) on an agar plate that contains LB medium supplemented with ampicillin and Xgal (LB + amp+Xgal), what result do you expect to obtain and why?

Cloning 2

Below is the restriction map of a 10 kb piece of DNA. Also shown below is a cloning vector which has two unique restriction enzyme recognition sites, one for EcoRI (E) and one for HindIII (H). The location of the kanamycin (kan) and ampicillin (amp) resistance genes is also shown. Kanamycin and ampicillin are antibiotics that are commonly used to select transformed E. colicells (consult the Lab Manual for more information). Note that the HindIII site is located within the amp gene. The Ori region is not shown for simplicity.

In order to clone the 1.9 kb fragment of the DNA of interest into the vector, you performed the following experiments:

1. digested the DNA of interest with EcoRI and HindIII to retrieve the 1.9 kb fragment

2. digested the vector with the same enzymes

3. mixed the digested DNA and the digested vector together and ligated, thus you created a

   recombinant vector

4. transformed kanamycin-sensitive E. coli competent cells with the recombinant vector and

   grew in SOB medium

5. plated the transformants on an agar plate containing LB medium supplemented with

   kanamycin and Xgal (LB + kan + Xgal), and incubated the plate at 37C in order to select kanamycin-resistant white colonies

Question: If you plate the selected kanamycin-resistant transformants (step 5 above) on an agar plate that contains LB medium supplemented with ampicillin and Xgal (LB + amp +Xgal), what result do you expect to obtain and why?

Answer 1

When you will plate Kanamycin resistant white transformed colonies on amp LB-Amp plates then There will be no colonies on LB -ampicillin plates as the gene conferring  resistance to ampicillin has Hind III restriction site so while restriction digestion and cloning the insert , the site was acted upon by restriction enzyme thus disrupting the amp gene. Disruption of gene result in loss of amp resistance.