Biotech lab week 2-quiz on protocol-Day 3

Question 1

What is added initially to the restriction enzyme reaction before the addition of the enzyme?

Answer 1

pROK2 DNA, NeBuffer, ddH20

Question 2

Before the addition of the enzyme, but after the addition of the precursor reagents, what is done to collect all the liquid at the bottom?

Answer 2

Pipet together DNA, buffer and water and gently vortex on medium speed followed by a brief pulse spin.

Question 3

What should the total reaction volume be?

Answer 3

50 uL

Question 4

When adding the enzyme, it is important to minimize its exposure to?

Answer 4

Temperatures above -20C to prevent deactivation.

Question 5

The tubes for the restriction digest are incubate for how long and at what temperature?

Answer 5

37C and 1 hour

Question 6

What is done during the incubation time?

Answer 6

Preparation of a biotinylated probe

Question 7

What is the first step for the preparation of the biotinylated probe?

Answer 7

Thaw out all the non-enzymatic labeling components.

Question 8

What are the non-enzymatic labeling components?

Answer 8

cDNA template, 5X reaction buffer, biotin labeling mix, ddH2O

Question 9

After thawing the non-enzymatic labeling components, where are they placed?

Answer 9

on ice

Question 10

What is done to recover the full volume of the tubes?

Answer 10

Brief centrifugation

Question 11

How much DNA is added to the 1.5 mL microcentrifuge tube?

Answer 11

1 ug

Question 12

How much ddH20 (nuclease free water) is added to the DNA, to reach a total volume of 34 uL, if the DNA concentration is 50 ng/uL?

Answer 12

If the concentration is 50 ng/uL (similar to ours). (for the DNA), then since we added 1 uL of DNA, then:
1 uL/50 ng * 1000ng (required amount of template) = 20 uL.
34 -20 = 14 uL

Question 13

What is the required amount of template DNA?

Answer 13

1000 ng

Question 14

What is the volume for the reaction with DNA for the making of the probe?

Answer 14

34 uL

Question 15

After adding the water to the DNA, what is added?

Answer 15

10 uL of 5X nucleotide reaction buffer.

Question 16

Once the nucleotide reaction buffer is added, what is done to the tube?

Answer 16

Vortex and pulse centrifuge to collect all the liquid at the bottom of the tube.

Question 17

What conditions are used to denature the template?

Answer 17

95C on a dry block for 7 minutes, then place on ice for 3 minutes (Briefly centrifuge)

Question 18

After denaturing the template, what is added?

Answer 18

5 uL of biotin labeling mix and 1 uL of Klenow reagent (to be added by the GA)

Question 19

How long is the template biotinylated for?

Answer 19

37C incubator overnight

Question 20

After the 1 hour incubation, for the restriction digest, what is done?

Answer 20

add 5 uL of 6x DNA loading dye and a brief spin. The tubes are then stored.

Question 21

What is the purpose of adding the 6x DNA loading dye?

Answer 21

terminate the reaction, this dissociates the enzyme from the DNA (NEB tools)