Laboratory 3 Flashcards
What was the set up for this exeperiment?
We retained the pellets from T0 to T90 from the previous practical
What must you first do to the pellets retained from last week?
They must be re-supsended in 450 ul of SDS sample buffer
What exactly are you doing in this practical?
Creating a gel to carry out electrophoresis of your samples from T0 to T90
Why do you not need to add size marker to your samples?
Size marker is already in the sample buffer
Write a note on your size marker
The protein markers are pre-stained and have molecular weights of:
175
80
58
46
30
25
17
7kDa
How do we confirm protein expression of Green fluorescence protein?
By SDS page analysis
How do we confirm protein expression of Green fluorescence protein?
By SDS page analysis
Why do we use SDS page analysis to confirm protein expression?
It allows for visualisation of expressed proteins and allows for an estimation of the molecular weight of the protein
What gels do you need to prepare in this experiment?
A 12% resolving gel
A 5% stacking gel
What gel system do we use?
A mini ATTO gel system
What do you need to prepare our resolving gel?
(5)
H2O
30% acrylamide mix
1.5M Tris Cl pH8.8
10% SDS
10% ammonium persulphate
TEMED
How should you pour the resolving gel?
Pour into gel template up to approximately 1.5cm from the top of the notched plate
What should you add after the resolving gel?
(2)
0.1% SDS solution
and leave to set for at least 20 minutes
What is the second gel you have to prepare?
3ml of the 5% stacking gel
What do you need to make the stacking gel?
(6)
H2O
30% acrylamide mix
1M Tris Cl pH6.8
10% SDS
10% ammonium persulphate
TEMED
What should you do before adding the stacking gel?
Pour of the 0.1% SDS overlay
What should you do after adding your 3ml of stacking gel?
Insert the comb into the template and allow the gel to set for 20 mins
How do you set up the gel?
(4)
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
Why must you wash the wells with buffer?
TO remove unpolymerised acrylamide
How should you go about electrophoresing the gel?
(2)
Electrophorese at 125V/100mA until the dye has reached the bottom of the gel
This should take approx 45 minutes
What dye should you use in your samples?
Bromophenol Blue Dye
What should you do with the gel after electrophoresis?
(3)
Remove it with a scalpel
Transfer resolving gel to staining solution containing 1% Commaissie Blue Stain
After 1 hour, de stain for several hours
