Laboratory 3

Question 1

What was the set up for this exeperiment?

Answer 1

We retained the pellets from T0 to T90 from the previous practical

Question 2

What must you first do to the pellets retained from last week?

Answer 2

They must be re-supsended in 450 ul of SDS sample buffer

Question 3

What exactly are you doing in this practical?

Answer 3

Creating a gel to carry out electrophoresis of your samples from T0 to T90

Question 4

Why do you not need to add size marker to your samples?

Answer 4

Size marker is already in the sample buffer

Question 5

Write a note on your size marker

Answer 5

The protein markers are pre-stained and have molecular weights of:
175
80
58
46
30
25
17
7kDa

Question 6

How do we confirm protein expression of Green fluorescence protein?

Answer 6

By SDS page analysis

Question 7

How do we confirm protein expression of Green fluorescence protein?

Answer 7

By SDS page analysis

Question 8

Why do we use SDS page analysis to confirm protein expression?

Answer 8

It allows for visualisation of expressed proteins and allows for an estimation of the molecular weight of the protein

Question 9

What gels do you need to prepare in this experiment?

Answer 9

A 12% resolving gel

A 5% stacking gel

Question 10

What gel system do we use?

Answer 10

A mini ATTO gel system

Question 11

What do you need to prepare our resolving gel?
(5)

Answer 11

H2O
30% acrylamide mix
1.5M Tris Cl pH8.8
10% SDS
10% ammonium persulphate
TEMED

Question 12

How should you pour the resolving gel?

Answer 12

Pour into gel template up to approximately 1.5cm from the top of the notched plate

Question 13

What should you add after the resolving gel?
(2)

Answer 13

0.1% SDS solution

and leave to set for at least 20 minutes

Question 14

What is the second gel you have to prepare?

Answer 14

3ml of the 5% stacking gel

Question 15

What do you need to make the stacking gel?
(6)

Answer 15

H2O
30% acrylamide mix
1M Tris Cl pH6.8
10% SDS
10% ammonium persulphate
TEMED

Question 16

What should you do before adding the stacking gel?

Answer 16

Pour of the 0.1% SDS overlay

Question 17

What should you do after adding your 3ml of stacking gel?

Answer 17

Insert the comb into the template and allow the gel to set for 20 mins

Question 18

How do you set up the gel?
(4)

Answer 18

Remove the comb and gasket

Insert the gel rig with the notched plate facing the inner chamber

Fill inner and outer buffer chambers with tris-glycine

Wash wells with tris-glycine

Question 19

Why must you wash the wells with buffer?

Answer 19

TO remove unpolymerised acrylamide

Question 20

How should you go about electrophoresing the gel?
(2)

Answer 20

Electrophorese at 125V/100mA until the dye has reached the bottom of the gel

This should take approx 45 minutes

Question 21

What dye should you use in your samples?

Answer 21

Bromophenol Blue Dye

Question 22

What should you do with the gel after electrophoresis?
(3)

Answer 22

Remove it with a scalpel

Transfer resolving gel to staining solution containing 1% Commaissie Blue Stain

After 1 hour, de stain for several hours