yeast reagent practice

Question 1

10X Fastdigest Buffer + FastDigest EcoRI

Answer 1

restriction enzyme digest

Question 2

TE/SDS

Answer 2

SDS helps to break down the yeast cell wall

Question 3

Digest solution

Answer 3

used to resuspend the yeast cells, provides appropriate conditions for Proteinase K

Question 4

Proteinase K

Answer 4

digests any proteins that are present

Question 5

RNase A solution

Answer 5

removes RNA

Question 6

lysis solution

Answer 6

contains components that will ensure complete lysis of cells. It also contains chaotropic salts and helps maintain the right pH for binding of the DNA to the silica membrane on the column

Question 7

70% ethanol

Answer 7

facilitates the binding of the DNA to the silica membrane when the prepared lysate is added to the column

Question 8

wash buffers

Answer 8

remove impurities from the DNA bound to the membrane of the column

Question 9

elution buffer

Answer 9

causes purified genomic DNA to come off the membrane of the column

Question 10

alkaline phosphatase

Answer 10

catalyzes the release of 5’ phosphate groups from DNA, which prevents recircularization of the vector during ligation

Question 11

T4 DNA ligase

Answer 11

makes recombinant plasmid

Question 12

T solution

Answer 12

makes bacterial cells competent for transformation

Question 13

alkaline lysis solution 1

Answer 13

glucose: maintains osmolarity

Tris: buffers the solution, allowing cells to be resuspended in an isotonic solution

EDTA: chelating agent that forms strong complexes with divalent metal ions. This inactivates DNases present and destabilizes the membrane

RNase A: remove RNA

Question 14

alkaline lysis solution 2

Answer 14

SDS: dissolved components of the membrane and denatures cytoplasmic proteins

NaOH: helps break up the cell wall thereby freeing cellular components into the solution, and it denatures DNA

Question 15

alkaline lysis solution 3

Answer 15

KOAc: drops the pH dramatically (neutral), causes DNA and proteins to aggregate and precipitate. KOAc interacts with SDS and the complex precipitates. Neutral pH=DNA can renature properly

Question 16

ethanol + KOAc

Answer 16

used to precipitate the plasmid DNA

Question 17

70% ethanol

Answer 17

removes residual salt from the already precipitated plasmid DNA