Biotechnology (W16) - Week 3,4 - Day 5 Protocol: Denaturation of an agarose gel

Question 1

Prior to transfer and hybridization, the agarose gel needs to have what done to it?

Answer 1

Needs to be exposed to an acid bath and a subsequent alkaline wash.

Question 2

What is the purpose of the acid bath?

Answer 2

Required to depurinate the DNA strands such that larger fragments greater than 15 kb are cleaved and thus transfer more readily to the membrane.

Question 3

What is the purpose of the alkaline solution?

Answer 3

Breaks the hydrogen bonds between the two-strands of separated DNA fragments. The fragments then become single-stranded, thus allowing for the ss biotin-labeled probe to bp with its complementary DNA in the upcoming hybridization step.

Question 4

To determine the orientation of the gel later in the lab, what is done?

Answer 4

Use a razor blade to cut a small piece of the gel from the bottom left hand corner of the gel.

Question 5

After removing part of the gel for orientation, where is it placed, and what is done?

Answer 5

Place the gel carefully in a shallow tray containing 200 mL of distilled water. Place the tray on a shaker platform for 3 minutes. After this, using gloves, hold the gel in place and remove the water into the sink.

Question 6

After removing the water, what is done?

Answer 6

Add 200mL of acid wash to the gel. Place on shaker, set a low speed and rock for 15 minutes.

Question 7

During the acid wash, what may happen?

Answer 7

The blue dyes may change to yellow or green

Question 8

After the acid wash, what is done?

Answer 8

Decant the acid and refill with 200 mL of distilled water. Rock for 5 minutes.

Question 9

After the second water wash, what is done?

Answer 9

Decant water, add alkaline solution and rock for 30 minutes.

Question 10

During the alkaline wash, what is done?

Answer 10

Prepare the capillary transfer setup. (i.e. obtain the chromatography/filter paper, paper towels, blotting paper and support.

Question 11

Both the alkali solution and acid solution are decanted into what?

Answer 11

Their specific waste containers.

Question 12

After decanting the alkali solution, what is done?

Answer 12

Refill tray with 200mL distilled water and rock for 5 minutes.

Question 13

After decanting the water following the alkaline wash, what is done?

Answer 13

Add 200mL of neutralization buffer and rock for 15 minutes.

Question 14

While doing the neutralization buffer thing, what do you do?

Answer 14

Prepare the membrane for transfer.

Question 15

Why is it important to use appropriate gloves and blunt-end forceps?

Answer 15

A membrane that has been touched by oily hands will not hand.

Question 16

What corner is marked on the gelerino no copypastarino?

Answer 16

the bottom left, with pencil, initials.

Question 17

The membrane is placed where at the start of transfer?

Answer 17

In a small container with 10-20 mL of water.

Question 18

The membrane must be submerged for how long?

Answer 18

30 seconds

Question 19

After the membrane has been submerged in water, what is done?

Answer 19

Pour off water into the sink, and then add 50 mL of 10X SSC. Rock for 15 minutes!!!!!

Question 20

After the neutralization buffer wash is complete, what is done?

Answer 20

Discard buffer into the dishes sink and add 200mL distilled water. Rinse for 5 minutes.