Biotech W16-Lab day 4-week 2-protocol quiz

Question 1

For the determination of biotin labeling efficiency, what are samples A, B and C?

Answer 1

A - commercially prepared biotin-labeled DNA
B - control reaction made for us using the same buffers and enzymes
C - the student prepared biotin-labeled probe

Question 2

What sample is kept (between A, B and C)?

Answer 2

C

Question 3

What are the dilutions that are made for each of the samples?

Answer 3

1: 10
1: 100
1: 500

Question 4

What is the commercially prepared biotin labeled DNA and how is it diluted?

Answer 4

Fermentas DNA. Add 1 uL of it and 9 uL of water for the first dilution.

Question 5

What is the test blot membrane made of?

Answer 5

nylon

Question 6

What is important when handling the membrane?

Answer 6

wear gloves and only touch the edges.

Question 7

What volume of each of the diluted samples is added to the membrane?

Answer 7

1 uL

Question 8

How many grids should there be, and how are these drawn, on the membrane?

Answer 8

12 and with a permanent marker

Question 9

How long is the test blot allowed to dry?

Answer 9

15 minutes and air dried

Question 10

What is done to ensure that the DNA samples are not washed off the membrane?

Answer 10

Cross-linking of the DNA samples to the GeneScreen Plus membrane

Question 11

What is the desired program in the GS gene linker?

Answer 11

(C-L: 125 ms)

Question 12

The membrane is placed on a paper towel and placed where?

Answer 12

in the Bio-Rad GS gene linker

Question 13

AT the end of the program, what will be heard (i.e. after reaching the set time of the set energy level)?

Answer 13

A tone for a few seconds

Question 14

How can the tone be stopped?

Answer 14

By pressing any button

Question 15

To begin the chemiluminescence reaction (i.e. to get it prepped, not run it), what is done first?

Answer 15

Add 10 mL of blocking solution too a hybridization bottle.

Question 16

What is the blocking solution?

Answer 16

1X TBST + 1% skim milk

Question 17

What is the purpose of the blocking solution?

Answer 17

The blocking solution utilizes a inert protein to prevent non-specific interaction of the antibodies.

Question 18

How is the test strip transferred to the tube?

Answer 18

Using clean forceps.

Question 19

How long is the strip with the washing solution added to the rotisserie (fkn top kek)?

Answer 19

15 minutes at room temperature

Question 20

How is the membrane washed?

Answer 20

Decant the blocking solution and wash the membrane with 10 ml of 1X TBST. Allow to wash for 5 minutes.

Question 21

During the first wash, what is done?

Answer 21

Prepare stabilized streptavidin peroxidase conjugate.

Question 22

How is the stabilized streptavidin peroxidase conjugate prepared?

Answer 22

Add 10 uL of streptavidin peroxidase conjugate to a 10 mL fresh aliquot of 1X TBST + 1% skim milk and vortex for 2 seconds to mix.

Question 23

After decanting the wash buffer, what is added to the test strip?

Answer 23

The streptavidin horseradish peroxidase conjugate.

Question 24

How long is the test strip and enzyme conjugate incubated for on the rotisserie?

Answer 24

25 minutes at room temperature

Question 25

What is the purpose of using streptavidin?

Answer 25

It has a very strong affinity for biotin (as you may have guessed)

Question 26

After the 25 minute incubation, what is done?

Answer 26

Pour of the conjugate solution and wash with 10 mL of TBST for 5 minutes. (bis = latin repeat)

Question 27

AFter the second washing step, what is done?

Answer 27

Remove the test strip from the buffer and place into a large weigh dish. add TBST

Question 28

What is the purpose of adding TBST after the wash step is complete?

Answer 28

Keep the membrane from drying out.

Question 29

How is the “light” image taken?

Answer 29

Place the test strip in the imager using forceps and acquire the image.

Question 30

What is done for the chemiluminescent reaction?

Answer 30

Do not move strip after taking “light” image.
Mix luminol and oxidizing agent, invert gently to mix but not make bubbles.
Pour the mix on top of the test strip and incubate for 1 minute.
Set the syngene imaging system to chemiluminescence and capture an image of the test strip with the overhead light off.