Miniprep

Question 1

What is transformation?

Answer 1

Process by which foreign DNA is introduced into a competent cell.

Question 2

What other gene is present in the E.Coli plasmid?

Answer 2

Kanamycin resistance

E.Coli cells are placed in a kanamycin supplemented LB broth, whcih

Question 3

What else does the LB broth contain, when growing transformed E.Coli Cells?

Answer 3

Kanamycin antibiotic is present. This means that only E.Coli cells with the plasmid (i.e. had been transformed) will grow (which also contains the DNA for green fluorescent protein (GFP)

Question 4

What are the two methods of transformation, and which one was utilized for E.Coli cells?

Answer 4

Chemical transformation and electrical transformation. Chemical transformation was used.

Question 5

With respect to miniprep, solution I contains glucose, EDTA and TRIS. What stage of miniprep does this refer to, and what function do each of these serve?

Answer 5

Re suspension of the cells after centrifuging.

• Glucose maintains osmotic pressure so the cells do not lyse early
• TRIS acts as a buffer
• EDTA chelates cations (e.g. Ca2+, Mg2+). DNAases require cations, so EDTA chelation prevents DNA degradation.

Question 6

With respect to miniprep, solution II contains NaOH and SDS. What stage of miniprep does this refer to, and what function do each of the components serve?

Answer 6

Lysis.
NaOH: denatures the hydrogen bonds between DNA, resulting in conversion from double stranded DNA to single stranded DNA. Also disrupts the cell wall
SDS: Detergent that disrupts the cell membrane, resulting in lysis

Question 7

With respect to miniprep, solution III contains K-acetate, glacial acetic acid and water. What stage of miniprep, and what function?

Answer 7

Neutralisation step.
K-acetate reduces alkalinity of solution, and allows hydrogen bonds to form on plasmid DNA. Chromosomal DNA however remains single stranded, as annealing can only occur on shorter strands.
Water: plasmid double stranded DNA can dissolve in water, whereas single stranded DNA can. So plasmid dissolves, and chromosomal DNA (single stranded) +protein+SDS is spun out as pellet :O

Question 8

With respect to miniprep, what is the final stage and what solution do you need

Answer 8

Final stage is alcohol precipitation.

Isopropynol cleans salts off, and precipitates plasmid DNA so it forms into a pellet after centrifuging