MOLECULAR BIOLOGY PRACTICAL

General Tips

● Skim through the whole practical. If there is any experiment that requires long waiting/incubation
time (e.g. agarose gel electrophoresis), start on that experiment first otherwise you may not be able
to finish.
● Practice pipetting with your main hand & opening & closing Eppendorf tubes with only your non-
main hand (saves time).
● Label all your Eppendorf tubes to avoid confusion.

● Cap back your box of pipette tips after each use to minimize contamination (your aseptic
techniques may be assessed).
● Balance the centrifuge with a blank tube of the same mass (may be assessed).

● For reagents, vortex them/pipette up & down to ensure a homogeneous mixture before
transferring to the main reaction mixture.
● For organisms (e.g. bacteria & yeast), invert the tube a few times to mix (do NOT vortex or pipette
up & down unless otherwise instructed otherwise you will kill or disrupt the chains of microbes
which you may be asked to observe later).
● If instructed to leave the tubes on ice, you must leave the tubes on ice otherwise important
enzymes may be denatured & lose their activity (especially important for restriction digestion).
● Be prepared for cool, new experiments which you have not done before. The principles behind
these experiments may be similar to those of experiments which are familiar to you.

Gel Electrophoresis

● To avoid shaking arms, rest your elbows on the table & support the barrel of the micropipette (the
narrow part just above the tip) with the finger of your other hand while loading.
● Ensure that your micropipette is down to the first stop as you are removing the tip from the well.
Just as your tip leaves the liquid surface, depress to the second stop to empty out all sample from
the tip. (Actually, you do NOT have to completely empty the sample from the micropipette tip when
loading the gel. Just empty the tip until you reach the first stop, then remove the tip from the well &
electrolyte while maintaining your hold on the first stop. This helps to avoid bubbles. ☺)
● To better visualize the wells, place a black sheet of paper or any patterned sheet of paper beneath
the electrolysis chamber for contrast. If no paper is provided, position your head directly above the
row of wells in the gel to better visualize the wells.
● If your samples float out of the wells for some unforeseen reason, load as much of the sample as
you can spare into the wells (not caring if it continues to float out) & run the gel as per normal. Try
to use the micropipette tip to push the floating sample away from the other wells in the gel to avoid
 cross-contamination of the samples. (If you do this, your gel should have at least some faint bands
upon visualization rather than not having electrophoretogram at all.)

Aseptic Techniques (Plating)

● Label the edge (NOT the middle) of the bottom of the plate.

● Clear the bench top around the burner so that you have an empty space. Wipe the bench top with
some ethanol to sterilize it.
● Place the ethanol bottle away from the burner. Then, uncap the burner & light a flame using the
lighter.
● Hold the culture plate right side up near the flame & open the lid slightly at a 45 degree angle with
the fingers of one hand. (Note that the culture plates may be given to you inverted as they are
usually stored inverted. Also, do NOT open the lid completely otherwise you will contaminate the
culture media.)
● Flame the metal inoculating loop until red hot. Allow the loop to cool by agitating the loop near the
flame before introducing into the inoculum (if the hot loop is used to collect the inoculum, you will
kill the bacteria).
● If the inoculating loop is a sterile plastic loop, do NOT flame it (ready to use).

● Using your other hand, insert the inoculating loop/pipette tip into the gap between the lid and the
plate & introduce the inoculum onto the culture media by streaking/spreading/pipetting out the
inoculum.
● Streaking to obtain individual colonies:

● Spreading to obtain bacterial lawn:

● After spreading the inoculum, allow the lid to cover the plate once again. Use parafilm to seal the
space between the lid & the plate: Peel off the paper strip from the parafilm. Use your thumb to
hold one end of the parafilm against the side of the plate. Use the other hand to stretch the parafilm
around the side of the plate. Slide your thumb further along the side of the stretched parafilm and
repeat stretching action until the parafilm is all the way around the plate. Stretch the free end of the
parafilm upwards & stick it to the top of the lid of the plate.
● You are finally done plating ☺. Extinguish the flame by capping the wick.

Revise theory: Agarose gel electrophoresis; bacterial transformation & recombinant DNA technology;
mechanisms of antibiotic action & disc diffusion test; SDS-PAGE for proteins; purification of RNA & DNA;
purpose of steps in procedure (!!!).