Lab 7 Flashcards
Buffer PB
enables binding of products to the spin-column membrane?
Buffer PE
wash buffer to remove contaminants
Buffer EB
elution buffer
pH indicator 1
changes the color of the solution to indicate whether or not the pH is too high
loading dye
makes the DNA solution much denser than the
electrophoresis buffer
dyes the DNA so it’s easy to see on the uv transilluminator
Buffer PN
promotes absorption of bases and fragments to the membrane
Buffer QG
solubilizes the agarose gel slice and provides the right conditions for binding DNA to the membrane
What does a yellow solution mean?
optimal pH
what does a purpose solution mean?
pH is too high
elution efficiency is strongly dependent on…
salt concentration
pH of the elution buffer
DNA yield depends on three factors:
the volume of elution buffer
how the buffer is applied to the column
the incubation time of the buffer on the column
Gel purification steps:
add QG buffer to gel slice
incubate 50 degrees until the gel slice is dissolved (you can vortex every few minutes to help this)
make sure the color is yellow
add isopropanol and mix
place into the spin column and centrifuge 1 minute
discard flow-through and add Buffer QG
centrifuge for 1 minute, discard flow through. add buffer PE
centrifuge for 1 minute. discard flow through
SPIN WITH NOTHING IN IT FOR 1 MINUTE
place into a clean microcentrifuge tube, add EB buffer and centrifuge for 1 minute
run a gel if you need to run a gel?
if the pH is too high, add…
sodium acetate
if the gel slice was too large…
use multiple columns
Conditions for DNA binding to membrane and for eluting DNA from the membrane
low salt concentrations
pH less than or equal to 7.5
