Insertion and Isolation of
Plasmid
Determining Protein
Concentration
SDS Gel Electrophoresis
Materials Used:
Laemmli sample buffer (dye)- which is made of 62.5 mM Tris-HCl, 25% glceryol,
2% SDS, 0.01% Bromophenol Blue
Gel Electrophoresis chamber
Solid lysozome
Solid hemo
$-mercaptoethanol
(kills the florescence)
TE Buffer
p-Glo sample
Procedure:
First we made Laemmli sample buffer, by adding 50:l
of $-mercaptoehanol
for the 950:l
sample buffer, so that the final concentration would be 5%
$-mercaptoethanol,
this was the dye.
We determined the amount of solid lysozome we would need to make a
sample that was 5g/l, which came out to be .0062g so we added that to 1mL of
TE buffer.
Then we diluted that down by taking 61:l
of the sample and adding it to 839:l
to bring the solution back to 1mL.
We determined how much solid hemoglobin we would need to make the
sample 1g/l, which came out to be .0057g and we did the same thing with
this, added 1mL of TE buffer to it.
Then we took 175:l
of that sample placed it in another tube and adding 825:l
of TE to it.
Next we took out 10:l
of out lysozome, hemoglobin and our p-Glo samples, placed them in different
tubes and added 3ml of dye to them.
We took our three samples plus a standard sample and placed them in
boiling water for 2 minutes.
We injected our samples into the chambers immediately after we
pulled them out of the water to prevent any damage to the sample.