Flashcards in Biotechnology (W16) - Week 3,4 - Day 5 Protocol: Denaturation of an agarose gel Deck (20)
Prior to transfer and hybridization, the agarose gel needs to have what done to it?
Needs to be exposed to an acid bath and a subsequent alkaline wash.
What is the purpose of the acid bath?
Required to depurinate the DNA strands such that larger fragments greater than 15 kb are cleaved and thus transfer more readily to the membrane.
What is the purpose of the alkaline solution?
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.
To determine the orientation of the gel later in the lab, what is done?
Use a razor blade to cut a small piece of the gel from the bottom left hand corner of the gel.
After removing part of the gel for orientation, where is it placed, and what is done?
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.
After removing the water, what is done?
Add 200mL of acid wash to the gel. Place on shaker, set a low speed and rock for 15 minutes.
During the acid wash, what may happen?
The blue dyes may change to yellow or green
After the acid wash, what is done?
Decant the acid and refill with 200 mL of distilled water. Rock for 5 minutes.
After the second water wash, what is done?
Decant water, add alkaline solution and rock for 30 minutes.
During the alkaline wash, what is done?
Prepare the capillary transfer setup. (i.e. obtain the chromatography/filter paper, paper towels, blotting paper and support.
Both the alkali solution and acid solution are decanted into what?
Their specific waste containers.
After decanting the alkali solution, what is done?
Refill tray with 200mL distilled water and rock for 5 minutes.
After decanting the water following the alkaline wash, what is done?
Add 200mL of neutralization buffer and rock for 15 minutes.
While doing the neutralization buffer thing, what do you do?
Prepare the membrane for transfer.
Why is it important to use appropriate gloves and blunt-end forceps?
A membrane that has been touched by oily hands will not hand.
What corner is marked on the gelerino no copypastarino?
the bottom left, with pencil, initials.
The membrane is placed where at the start of transfer?
In a small container with 10-20 mL of water.
The membrane must be submerged for how long?
After the membrane has been submerged in water, what is done?
Pour off water into the sink, and then add 50 mL of 10X SSC. Rock for 15 minutes!!!!!