Detection of Alu by PCR

WHS Independent Research Project 2011
Protocol: Growing up a liquid culture of your bacteria.
1. Be sure to put on gloves.
2. Without removing the lids from your plates (to avoid contamination), observe the bacteria growing
on each of your plates. You may use the microscopes to do this if you wish. In your lab notebook,
record what you see. How many obviously different kinds of bacteria are on each of your plates?
What do they each look like? Take note of differences in colony color, shape, consistency, etc. in your
notes. You should already be doing this as standard practice for your IRP.
3. Come to a consensus among your team members of which colonies you are going to analyze. Each
group will be performing all subsequent experiments on THREE colonies of your choice. Be sure to
discuss with your team which colonies to select so that you have the best chances of picking different
species from each other. To ensure that you are all selecting different species, take into consideration
the appearance of the colonies and their source. (For example, different sources are likely to have the
same bacteria if they have both been handled by human hands.)
4. Obtain enough 1.5-ml eppendorf tubes for each bacterial colony chosen by the group. Label each
with your initials and a word that reminds you from which source the bacterial sample came. Add 1ml
LB (Luria broth- essentially a liquid version of the nutrient agar we use) to each tube.
5. Please check in with Ms. Boehm before completing this step. Pick up a sterile stick or
inoculating loop from one end, being careful not to touch the bottom of the stick to any surface,
including your fingers. Dab up the bacteria from a single colony on your plate onto the bottom end of
your sterile stick. The colony you pick needs to be 2-3mm in size, and should not be touching any
other colony. (If you pick up bacteria from more than one colony on your stick, you will get a mix of
two DNA sequences that will not allow you to identify your species.)
6. Spin around the end of the stick in 1ml of LB so that the bacteria comes off the stick and into the
liquid. You can also scrape the bottom of the stick along the inside of the tube to make sure the
bacteria comes off. Change sticks/loops between samples.
7. Close the tube tightly and vortex the sample for 30 seconds. This step will break up the colony into
cells in the liquid.
8. Place the tube in the thermomixer to shake overnight (approximately 18 hours) at 600RPM. The
thermomixer is set at 37C.
9. After the overnight shaking, you should look at your eppendorf tube and make sure this step worked.
You should now see a very cloudy solution where before it was clear. Record results.
10. The samples will be refrigerated, if necessary, until the next steps are completed in class.
Protocol: Polymerase Chain Reaction (PCR) to make many copies of one gene
from the bacterial DNA.
1. Put on new gloves for this step. It is important to keep ALL tubes COLD during this entire lab
prep, or else the PCR will not work. The enzyme needs to be stored at a very cold temperature at all
times, right up until PCR, or else it will significantly lose its efficiency.
2. Obtain a 0.2 ml strip of 12 sterile PCR tubes. Label each tube 1-12 ON THE SIDE OF THE TUBE,
NOT THE TOP, that you are going to use for each of your unknown DNA samples. This coding
system should be kept track of in your lab notebook with each number corresponding to the source and
characteristics of each colony. Add 48 µl of the “Master Mix” to each of your 12 PCR tubes at this
point. 10 tubes are for your bacteria and 2 are for controls (described below) This mix is a
combination of nucleotides, buffers, salts, primers and a polymerase, all which help make PCR work
correctly. Again, remember to keep your tubes on ice.
3. Pipet 2 µl, BUT NO MORE, of each of your bacterial liquid cultures into the designated PCR
reaction tubes (ie. 2ul of culture 1 into tube 1, 2ul of culture 2 into tube 2, etc). Change tips every
time you pipet during these steps! At this point, your total volume in each tube should be 50 µl.
4. One person in the class should also prepare one PCR tube for the negative control (water only) and
another person should set up a tube for the positive control (i.e., E. coli liquid culture known to work
in this assay). To set up the controls, pipet 2ul µl of the correct liquid (sterile water or E. coli prep) into
the correct tubes. Keep your tubes on ice.
5. Seal on the caps for the tubes as tightly as possible. Visually inspect to ensure no gaps between cap
and tube. Be careful not to squeeze the sides of the tube-these special tubes have thin walls so that the
temperature of the thermocycler rapidly equilibrates with the temperature of the reaction mixture. But
this also makes them fragile! Invert and flick the tubes gently to mix them for a few seconds. Then
spin the samples for a few seconds in a small centrifuge that has a rotor in it that fits PCR tubes.
Quickly put the tubes back on ice.
8. The DNA is now ready for amplification in the PCR machine. Once all tubes are ready, Ms. Boehm
will place them in the PCR machine and start it running. The reaction will proceed at the following
temperatures for the following amounts of time:
1 cycle:
95˚C for 10 minutes (initial denature)
40 cycles:
95˚C for 30 sec (denature)
60˚C for 30 sec (anneal)
68˚C for 1 min (extend)
1 cycle:
68˚C for 10 minutes (final extension)
4˚C overnight
Protocol: Gel electrophoresis to determine the size and amount of DNA.
1. Plug your gel apparatus (the red plastic piece of equipment) into the wall at your lab station. Many
groups will be sharing gels for this step.
2. Unwrap a gel from its foil wrap, and set the gel into the apparatus by placing the right side of the
gel down into the red apparatus first, and then clicking the left side into place.
3. Warm your gel up by pressing and holding down the “30 minute” button for a few seconds, until the
green light starts flashing.
4. In two minutes, your gel will be done warming up, and it will beep to tell you so. To stop the
beeping, press the “30 minute button” briefly (i.e. do not hold the button down this time). You can
move on to the next step while your gel is warming up.
5. You will be given three tubes of liquid: Water, Blue Dye, and ladder. Perform the following steps
for each of the PCR reactions:
a) Remove 10 µl of your PCR reaction and put it into a clean and LABELED 1.5 ml
microcentrifuge/eppendorf tube. At this point you should also pipet 11µl of your PCR reaction
into an eppendorf tube that will be sent off for sequencing. Give your remaining PCR product and
the sequencing tube to Ms. Boehm immediately for storage until next session.
b) Add 10 µl of dilute blue dye to the 10µl of PCR product in the 1.5 ml microcentrifuge tube. Be
sure to change tips on your pipetman each time you reach into a new liquid, so that you don’t
contaminate any of your tubes with material from any of your other tubes.
c) Mix the sample by flicking the tube, and return all liquid to the bottom of the tube by briefly
spinning it in one of the small purple microfuges.
6. Remove the plastic comb that is sitting inside the holes at the top of your gel. This comb is
protecting 12 indentations, or “wells,” in the gel. These wells are where you will load your DNA
samples into the gel using your P20 pipetman.
7. Load your blue-dyed DNA samples into the lanes of your gel. You will have 20µl in each tube, and
you should load the whole sample. Be sure to record which lane contains which PCR product. If you
did a positive or a negative control, prepare this control as you did for your PCR product in step #5,
and load this control into a lane that is next to the lanes with your samples. Change tips between
loading each lane.
8. Then load all 20µl of the ladder in the next empty lane to the left of your samples.
Note: “Ladder” is a collection of pieces of DNA whose sizes are known. You can determine the sizes
of unknown pieces of DNA by comparing the unknown pieces of DNA to those pieces in the ladder.
To do this, you compare how far the unknown pieces of DNA traveled to how far the pieces in the
ladder traveled. To see what a ladder looks like when it has been run through a gel, obtain a picture of
the ladder used from Ms. Boehm.
9. Load 20 µl of water into every lane in your gel that doesn’t have a sample loaded into it already (i.e.
the rightmost lanes and the leftmost lanes of the gel).
10. Once all of the lanes of the gel have liquid in them (either a DNA sample or water), press the “30
minute button” briefly (i.e. do not hold the button down this time). The light will be green now but
should not be flashing. This means that electrical current is passing through your gel.
11. Your gel is now running for 30 minutes. You will be able to confirm this by observing the
movement of your blue-dyed DNA through the gel. If the dye has not begun to slowly move after a
few minutes, please alert Ms. Boehm.
12. Your gel will beep to tell you that it is done. When it is done, unplug it from the wall.
13. Remove your gel from the gel apparatus and bring the gel to Ms. Boehm to visualize the gel (using
a transilluminator) and take a photo of the gel.
References for procedure:
Tureene, C.Y., Tschetter, L., Wolfe, J. and Kabani, A. (2001) Necessity of Quality-Controlled 16S
rRNA Gene Sequence Databases: Identifying Nontuberculous Mycobacterium Species. J. Clin. Micro.
39: 3637-3648.
Emerson, J. A. & Ratner, D. I. (2006) From Bench Top to Presentation in Seven Days: DNA
Sequencing Research Projects for Summer Workshops or Undergraduate Teaching Labs. Dev. Biol.
295: 350.
Protocol: Setting up DNA sequencing reactions.
When you arrive in lab, you will be given the rest of the PCR product from the other day.
1. Be sure to put on gloves.
2. Spin your tubes in the centrifuge for a few seconds so that your entire sample goes to the bottom of
the tube.
3. You will now prepare your samples for sequencing. To begin, VERY CLEARLY write the
appropriate reaction number on CAP of each 1.5 mL eppendorf tube. Ms. Boehm will have a key to
keep track of which sequencing reaction is labeled with which number, so come see me to obtain your
reaction numbers.
4. Add 11 µl of each PCR product to the appropriate tube, then you will add 1 µl of forward primer to
each tube. Change pipet tips EVERY time!
5. Securely cap your tubes and give them to Ms. Boehm.
6. We will send your tubes to the Dana Farber Cancer Institute’s sequencing center in Boston. The
results should be available for us online within 24 hours!!!