Flashcards in Biotech lab 3-protocol Deck (24)
At the start of the lab, to make sure your plates are clean, what reagent is used?
Ethanol, then wiped clean with a Kim wipe.
What is used to fill the plates when in the frame and why?
Distilled water is used to check for leaks. The frame is then inverted and dried with a Kim wipe.
What is used to make the resolving gel solution?
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.
Where is the resolving gel solution put?
Using a pipette, the solution is put down the spacer into each sandwich to a level about 4 cm from the top.
Where is water added for polymerization to occur?
About 0.5 mL of water is added, at a 45 degree angle, at the top of the acrylamide next to both spacers.
How long does polymerization take?
What is done after polymerization?
Water is poured off and absorbed with a Kim wipe.
How is the stacking gel prepared?
15 mL tube, mix stacking gel solution.
Add 25 uL of ammonium persulphate and 5uL of TEMED.
What is done after the stacking gel is prepared?
Each sandwich is filled with the stacking gel solution and then a comb is inserted into each sandwich.
Why can't bubbles form?
Oxygen will inhibit polymerization, and bubbles will cause a local distortion in the gel surface at the bottom of the wells.
How long is the gel allowed to sit?
How is 1L of 1 x SDS running buffer made?
900 mL of distilled water with 100 mL of 10 x SDS running buffer in a 1 L flask with a stir bar inside.
How long is the 1L of 1 x SDS running buffer mixed for?
5 minutes on a magnetic stir plate.
What is each well rinsed with after removing the combs from the gel?
What is the inner chamber filled with?
What are the outer chambers filled with?
How are the samples resuspended?
The total cell culture uninduced and induced are resuspended in 50 uL of 1 x PBS. The induces sonicate lysate and pellet are not.
How are all the samples prepared for SDS analyis?
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.
How is the protein ladder pipetted?
Using p10 pipettor and 3 uL of it.
How are the samples pipetted?
Using a p20 and loading 20 uL of it.
What sample goes in each lane?
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
What is the black lead?
What is the voltage set to?