Lab 7: DNA cloning II

Question 1

you cut the mitochondrial control region insert (PCR product) and the pUC19 plasmid vector with the enzyme

Answer 1

HindIII

Question 2

you will be purifying the cut insert and vector using the

Answer 2

Qiagen QIAquick kit

Question 3

why it is necessary to purify DNA in between enzymatic reactions

Answer 3

contaminants like proteins, salts, and other reaction components can inhibit or interfere with subsequent enzymatic processes, leading to inaccurate or unreliable results

Question 4

will function to ligate the PCR product into the vector

Answer 4

ligation reaction

Question 5

ligation reaction is very simple in that it only requires the addition of what

Answer 5

DNA ligase in the presence of buffers, ions, etc.

Question 6

why is it possible that the insert could ligate into the vector in either orientation (forwards or reverse)

Answer 6

Because we are only using a single restriction enzyme (HindIII)

Question 7

why can either orientation (forwards or reverse) be problematic

Answer 7

promoter for the gene is naturally part of the vector and the start codon needs to be placed next to the promoter (rather than on the other side)

Question 8

how to get around this why can either orientation (forwards or reverse) be problematic

Answer 8

use different restriction enzymes on each side of the gene so that the gene can only be ligated in a single orientation

Question 9

Another problem that can arise when using a single restriction enzyme

Answer 9

the inserts have the ability to ligate with each other rather than with the vector.

As a result, it is possible to get a vector that receives 10 copies of insert in tandem (instead of one).

Question 10

how to prevent the inserts have the ability to ligate with each other rather than with the vector.

Answer 10

using two different restriction enzymes prevents insert-insert ligation and vector-vector ligation.

Question 11

A third issue that needs to be considered when setting up a ligation

Answer 11

is the amount of plasmid and insert being used.

Question 12

To achieve maximal rates of ligation, it is imperative to

Answer 12

add in roughly equal amounts of plasmid and insert rather than an abundance of one over another.

Question 13

Adding to much plasmid or too much insert can lead to

Answer 13

reduced levels of ligation.