Hi, my name is Dr. Stephanie Chen from the Biotechnology Program, and today we're going
to go through an interim lab.
As you can see, we're in the classroom today, so its going to be a little bit different
than your normal lab.
The first thing that you're going to want to do is find your lab station number - it
will be set up with everything that you and your lab partner will need to complete this
lab.
The only PPE we will be using today is the gloves, so make sure you get a pair in your
size.
Your lab station should already be set up with your three plates from the transformation
and ligation you did the week before, and your
new culture tubes.
You also want to make sure any personal beverages or food items are stored away, either at the
sides of the classroom or down by your feet so that they are not on
the same surface as our bacteria.
So the first thing you are going to want to do, is count all the colonies on your experimental
plates from last week.
There are three main types of colony plates that you will encounter.
So the first type of plate you will encounter is a TNTC or "Too Numerous to Count".
This is when theres just way too many colonies to count,
especially when they are touching each other, and it is just impossible to determine exactly
how many there are.
So this plate would be given a designation of "TNTC" (Too Numerous to Count)
The second type of plate is when there are a lot of colonies that could probably be counted,
but it would take you a while to count all of them.
In that case, we do something called "quadranting" - you basically just draw a cross across the
plate, and you only count one fourth of the colonies.
Then you multiply that by four, to get an estimate of how many colonies are on the plate.
The third type of plate is where all the colonies can be fairly easily counted, and so you would
just go ahead and count this plate.
One easy way to keep track of the colonies when you are counting would be to use your
sharpie and just dot each colony as you count so that you wont lose
track.
The next step is going to be starting liquid cultures - and for this you want to make sure
that you only use your experimental plate - so
pick out the one with your ligation and set aside the other two plates.
For this you're going to use the toothpicks - you're going to take one out (be careful
to be sterile), pick one colony, and then drop it in one tube of liquid culture.
As you can see, there's six liquid culture tubes, so five are going to be your experimental
colonies, and the sixth is going to be from a positive control plate provided by
your instructor
Go ahead and get a toothpick, and then you want to pick colonies that are fairly isolated
and just streak across the top of the agar through
the colony, and then take that and drop it right in the culture tube.
Next we want to make sure we also incolutate a positive control - so for that, similarly
you're going to use your toothpick, pick a colony, and then drop that into one of your
six culture tubes as well
We only have culture tubes for five of our experimental colonies - make sure you label
all of your culture tubes with your station number,
and whether they're experimental or positive control
This will make sure your cultures are returned to you properly during your next lab.
So these liquid cultures will be used in your next lab session to purify out the plasmid
and do a restriction mapping experiment to see how many contain the Campari
insert that we want
Once you're done, all of your materials are going to need to go back on the cart that
should be at the front of the classroom - your three experimental plates from last week should
be taped back together.
After putting all your materials away, you will want to get out a bottle of ethanol and
paper towels and wipe down your desk area
The last thing will be to remove your gloves and put them in the biohazard so we can begin
class