Alkaline Extraction

Question 1

STEP 1 of Alkaline Extraction

Answer 1

Centrifuge broth cultures A and B for 1 minute in the microcentrifuge (to pellet the bacterial cells)

Question 2

STEP 2 of Alkaline Extraction (after centrifuging)

Answer 2

Pour off the broth supernatant, and invert onto tissue paper to remove any remaining liquid

Question 3

STEP 3 of Alkaline Extraction (after removing supernatant)

Answer 3

Use an automatic pipette to add ice-cold Solution 1 to the pellet, dislodge the pellet using the end of the pipette tip and resuspend

Question 4

What is Solution 1 (components and functions)?

Answer 4

Glucose - increases osmotic pressure outside the cells to make them vulnerable to rupture
Tris - buffering agent to maintain pH
EDTA - binds to ions that are required by DNAases, to inhibit them from degrading DNA

Question 5

STEP 4 of Alkaline Extraction (after adding Solution 1)

Answer 5

Add Solution 2 to the pellet-S1 mixture, and invert 5 times to mix; the solution should clear as the cells lyse

Question 6

What is Solution 2 (components and functions)?

Answer 6

Sodium hydroxide - alkaline, so ruptures the cells (also denatures DNA into single strands)
SDS (a detergent) - breaks up lipid membranes of cells, and solubilises cellular proteins

Question 7

STEP 5 of Alkaline Extraction (after adding Solution 2)

Answer 7

Add ice-cold Solution 3, invert 5 times and leave the tubes on ice for 5 minutes; white precipitate should appear

Question 8

What is Solution 3 (components and functions)?

Answer 8

Acetic acid - neutralises pH, allowing DNA to renature
Potassium acetate - precipitates SDS, cellular debris and chromosomal DNA from solution

Question 9

STEP 6 of Alkaline Extraction (after adding Solution 3)

Answer 9

Centrifuge for 5 minutes at high speed, to pellet the cellular debris

Question 10

STEP 7 of Alkaline Extraction (after centrifuging again)

Answer 10

Supernatant is transferred to another microcentrifuge tube using a pipette (avoiding the white precipitate)

Question 11

STEP 8 of Alkaline Extraction (after transferring supernatant)

Answer 11

Fill the tube with iso-propanol, invert to mix, and incubate at room temperature for 2 minutes (no longer!)

Question 12

Function of iso-propanol (and why only 2 minutes)?

Answer 12

It precipitates any nucleic acids (DNA and RNA) from solution - given time, it would also precipitate proteins

Question 13

STEP 9 of Alkaline Extraction (after adding IPA)

Answer 13

Centrifuge again for 5 minutes - nucleic acid MAY be visible as small pellet at the bottom of the tube - supernatant is removed with a pipette tip and discarded

Question 14

STEP 10 of Alkaline Extraction (after centrifuging AGAIN)

Answer 14

Ice-cold 70% ethanol is added; invert once and centrifuge for 1 minute

Question 15

Function of adding ethanol in STEP 10?

Answer 15

Ethanol helps to remove any remaining salts or SDS

Question 16

STEP 11 of Alkaline Extraction (after adding ethanol)

Answer 16

Remove ALL supernatant using a tip; a tissue may be used to remove visible ethanol (if careful); pellet is allowed to dry at room temperature; make sure ALL ethanol is removed, flick the tube to check

Question 17

STEP 12 of Alkaline Extraction (after removing ALL ethanol)

Answer 17

When the pellet is dry (after about 10 mins) resuspend in TE buffer, and flick the tube to help dissolve the DNA pellet

Question 18

Components and functions of TE buffer in STEP 12?

Answer 18

Tris - buffering agent to maintain pH
EDTA - binds to ions that are required by DNAases, to inhibit them from degrading DNA