Biotechnology (W16) - Week 3,4 - Day 5 Protocol: Gel electrophoresis of restriction digested genomic DNA fragments

Question 1

What does the gel contain?

Answer 1

Ethidium bromide

Question 2

Before running the gel, it must be submerged in what?

Answer 2

1 X TAE running buffer (3-6mm above the gel)

Question 3

Once receiving the restriction gDNA digests and the pROK2 restriction digest sample, what must be done?

Answer 3

Thaw at room temperature. Briefly vortex sample and pulse centrifuge.

Question 4

What volume of the gDNA samples are added to the gel?

Answer 4

35 uL

Question 5

What volume is added for the ladder?

Answer 5

5 uL

Question 6

What is the volume added for the controls?

Answer 6

20 uL

Question 7

What is the positive probe control?

Answer 7

500pg which will indicated the efficiency of the labeled probe. Will not be seen on the gel image but will be seen on the southern blot.

Question 8

On the southern blot, what band size will the positive probe control be?

Answer 8

556 bp

Question 9

How are the DNA fragments separated?

Answer 9

By gel electrophoresis through a 0.8% agarose gel. 100V. For about an hour.

Question 10

When running the gel, how long should it run?

Answer 10

until the purple band almost reaches the bottom of the gel

Question 11

Once the DNA has migrated sufficiently, what is done?

Answer 11

Take a picture of it in the SynGene Imager with a UV ruler lined up to lane 1 with the 0 cm mark at the wells.