Please explain the answers provided for the 4 questions asked. 1. (4 pts) In the
ID: 198119 • Letter: P
Question
Please explain the answers provided for the 4 questions asked.
1. (4 pts) In the cloning lab a) describe the selection method used and explain what type of colonies it allowed you to identify, and b) describe the screening method used and explain what type of colonies it allowed you to identify 2, (4 pts) The transformation efficiency (# of transformants obtained per ug of DNA) can be increased by using cells made competent to take up DNA. Give two ways in which the cells you used in the cloning experiment were handled to increase their competency? Give the rationale for how these treatments increase competency. 3. (3 pts) Suppose your negative control plates from the cloning experiment had lots of colonies, what would you conclude about your cloning experiment? Explain your reasoning. 4. (3 pts) In the Amalysis of Transformants Lab explain why multiple bands were seen in your lanes of isolated plasmid DNA even though we didn't cut the DNA before loading onto the gel. (Assume each lane had one plasmid only.) What accounts for the different bands seen in a lane?Explanation / Answer
1.a) The selection method used in the experiment involves plating the cells on media containing the antibiotic present in the vector, ampicillin. This would help in the identification of the transformed cells as they would have developed anitbiotic resistance.
b) The screening method used is blue/white selection of the colonies based on the insertional inactivation of -galactosidase gene. Colonies plated on media containing antibiotic, X-gal and IPTG will form blue and white colonies based on the negative and positive transformation respectively. In this screening technique, positive and negative colonies can be identified. Negative (non-recombinant) cells will produce -galactosidase that hydrolyzes the X-gal to give blue color. Transformed (positive) colonies will appear white in color as they donot synthesize the enzyme to hydrolyze X-gal.
2. To increase the transformation efficiency, cells were made competent using CaCl2 as the presence of Ca2+, a divalent cation, will increase the DNA uptake and also by creating a thermal imbalance across the cells by exposing them to a temperature of 42o, heat shock treatment. Calcium ions attract the negatively charged DNA and the lipopolysaccharide in the cell membrane forming a complex. DNA cannot enter the cell by crossing the cell membrane itself. The heat shock treatment depolarizes the membrane potential thereby lowering the negativity ultimately allowing the DNA to enter inside the cell.
3. Ideally, the negative control plates should not have any colonies as they would not confer anitbiotic resistance. Presence of high number of colonies in the negative control plate could indicate the presence of undigested vector along with the digested vector. It can also be due to re-ligation of the vector. If the vector has sticky ends, self-ligation would occur. There are chances of problems associated with the antibiotic used such as expired ones or incorrect concentration. This would lead to the growth of bacteria with or without antibiotic resistance.
4. The major reason for the presence of multiple bands in the lane could be due to nicked, linear, supercoiled and circular single-stranded DNA. The nicked DNA is a relaxed circular plasmid migrating the slowest followed by linear DNA. The circular single stranded DNA could be due to the permanent denaturation of the DNA during alkaline lysis. RNA contamination, if any, can be seen in the gel towards the end as a smear which could be due to insufficient concentration of the RNase used.