Lab 3 – Gel electrophoresis and sequencing Flashcards
The size of DNA fragments (and PCR amplicons) are commonly visualized and determined by
gel electrophoresis
is a technique used to separate charged molecules (in this case DNA, but also is used for RNA and protein
gel electrophoresis
will be located closer to the sample wells at the top of the gel,
heavier molecules (larger DNA or protein fragments)
will migrate further down the gel towards the positive electrode
lighter molecules
highly purified derivative of agar, and comes in a powdered form
Agarose
used to separate DNA fragments
Agarose
The agarose is dissolved in
same running buffer used for electrophoresis
Upon melting the agarose in this buffer (via the microwave or Bunsen burner), the molten agarose is poured into
a casting tray that contains a plastic “comb”
When poured in, the agarose will solidify around the
the “teeth” of the comb
The comb is removed following
solidification
The buffer we will be using for electrophoresis
Tris-Borate-EDTA (TBE)
TBE is highly buffered to maintain what pH
the pH at 8
what is the gel made of
buffer will be mixed with agarose
The concentration of the stock solution of TBE is
10X.
what concentration stock of TBE are we using
diluted 0.5X TBE solution
TAE is not as highly buffered and is less satisfactory for electrophoresis at high voltage
Tris/acetate/EDTA (TAE).
When a DNA fragment is to be isolated from a gel, however, yields are better with
TAE buffer
Before DNA is added to the wells in the solid agarose gel, it has to be mixed with a
loading dye
