OWL The Otter SGC-1, Руководство пользователя

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echnical Tips
T
SECTION 3
9
The difference between a well comb and a sharktooth
comb:
A well comb has rectangular teeth and is simi-
lar to combs used for protein gels. After
polymerization, these combs must be removed
carefully to avoid destroying the acrylamide
walls that create the wells. A sharktooth comb
has pointed teeth. It is placed with the flat side
down during gel casting to create a flat surface on the top of the gel and
reinserted with the teeth down towards the gel for loading. Samples are
loaded in the space between the teeth.
Sharktooth comb: Carefully insert the flat edge of the sharktooth comb
between the plates to a depth of 2 to 3mm below the shorter plate. If the
flat edge of the comb is inserted too deeply (more than 4 or 5mm) during
gel casting the resulting trough will be too deep to load samples easily
with a pipet. It is wise to make marks on the comb to distinguish the
right/left sides so the comb can be put back in the same orientation for
sample loading later, in the event that the material has any slight variation
in thickness. Place binder clamps over the comb and glass plates along the
upper edge to force the glass plates against the comb. This will ensure a
tight fit of the comb during and after polymerization. If top binder clamps
are not used the comb may not be tight enough to prevent sample leaks
between wells following gel polymerization. Do not clamp the outer edges
over the side spacers. Allow the gel to completely polymerize.
After polymerization, wash out the trough with buffer gently in order to
remove any unpolymerized acrylamide and excess urea. Place the comb
between the glass plates in the correct left to right orientation (the same
orientation the trough was cast in) with the comb teeth pointing down
toward the gel. Slowly and carefully slide the comb until it just makes
contact with the gel surface, without piercing it.
Well comb: Carefully insert the comb. Place binder clamps over the
comb and glass plates along the upper edge to force the glass plates against
the comb. After polymerization, flood the comb with 1X TBE and remove
gently. Rinse the wells out with deionized water or TBE in order to
remove excess urea or unpolymerized acrylamide.