Biotech lab 3-protocol

Question 1

At the start of the lab, to make sure your plates are clean, what reagent is used?

Answer 1

Ethanol, then wiped clean with a Kim wipe.

Question 2

What is used to fill the plates when in the frame and why?

Answer 2

Distilled water is used to check for leaks. The frame is then inverted and dried with a Kim wipe.

Question 3

What is used to make the resolving gel solution?

Answer 3

In a 15mL tube, 5 mL of resolving gel solution is mixed without the ammonium persulphate and the TEMED.
Then, the 15 mL tube is capped and swirled gently as to not make bubbles.
Then, 50 uL of 10% ammonium persuphate and 5 uL of TEMED are added, the tube is capped then swirled to mix.

Question 4

Where is the resolving gel solution put?

Answer 4

Using a pipette, the solution is put down the spacer into each sandwich to a level about 4 cm from the top.

Question 5

Where is water added for polymerization to occur?

Answer 5

About 0.5 mL of water is added, at a 45 degree angle, at the top of the acrylamide next to both spacers.

Question 6

How long does polymerization take?

Answer 6

30 minutes.

Question 7

What is done after polymerization?

Answer 7

Water is poured off and absorbed with a Kim wipe.

Question 8

How is the stacking gel prepared?

Answer 8

15 mL tube, mix stacking gel solution.

Add 25 uL of ammonium persulphate and 5uL of TEMED.

Question 9

What is done after the stacking gel is prepared?

Answer 9

Each sandwich is filled with the stacking gel solution and then a comb is inserted into each sandwich.

Question 10

Why can’t bubbles form?

Answer 10

Oxygen will inhibit polymerization, and bubbles will cause a local distortion in the gel surface at the bottom of the wells.

Question 11

How long is the gel allowed to sit?

Answer 11

30 minutes

Question 12

How is 1L of 1 x SDS running buffer made?

Answer 12

900 mL of distilled water with 100 mL of 10 x SDS running buffer in a 1 L flask with a stir bar inside.

Question 13

How long is the 1L of 1 x SDS running buffer mixed for?

Answer 13

5 minutes on a magnetic stir plate.

Question 14

What is each well rinsed with after removing the combs from the gel?

Answer 14

Tank buffer.

Question 15

What is the inner chamber filled with?

Answer 15

Tank buffer

Question 16

What are the outer chambers filled with?

Answer 16

Tank buffer

Question 17

How are the samples resuspended?

Answer 17

The total cell culture uninduced and induced are resuspended in 50 uL of 1 x PBS. The induces sonicate lysate and pellet are not.

Question 18

How are all the samples prepared for SDS analyis?

Answer 18

20 uL of sample and 20 uL of 6x SDS loading dye are combined and the tubes are placed in a 100 degrees C dry bath for 6-8 minutes. Then they are briefly pulse centrifuged.

Question 19

How is the protein ladder pipetted?

Answer 19

Using p10 pipettor and 3 uL of it.

Question 20

How are the samples pipetted?

Answer 20

Using a p20 and loading 20 uL of it.

Question 21

What sample goes in each lane?

Answer 21

1- Protein ladder (3uL)
2- 3 ul of 6x SDS loading dye
3- Total cell culture uninduced
4- 3 ul of 6x SDS loading dye
5- Total cell culture induced
6- 3 ul of 6x SDS loading dye
7- Induced sonicate lysate
8- 3 ul of 6x SDS loading dye
9- Induced sonicate cell pellet
10- 3 ul of 6x SDS loading dye

Question 22

What is the black lead?

Answer 22

Cathode

Question 23

What is the voltage set to?

Answer 23

140V

Question 24

When is the power supply turned off?

Answer 24

When the lead dye reaches the bottom of the gel.