PCR Lab

Purpose:  

We are doing this experiment to teach you more about how to run a successful PCR.  We also want to show you how one can determine if a DNA sample is bacterial by amplifying the 16S gene which amplifies at 1500 bp. The 16S gene is unique to the domain Eubacteria. If you do not see the 16S gene amplified on the gel then it may be due to contamination from non Eubacteria DNA or from human error.

 

~Introduction and PCR Overview~

What is PCR?

Polymerase Chain Reaction (PCR) is a process that uses primers to amplify specific cloned or genomic DNA sequences with the help of a very unique enzyme. This PCR process, invented by Kary Mullis in 1984, has been automated for routine use in laboratories worldwide (White). This procedure is so common that, once you have a piece of template DNA with the target sequence you need to amplify, you can just add the following three items:

 

1)      A pair of primers that hybridize (stick) to the beginning and end of the target

2)      All four dexoyribonucleate triphosphates (dNTPs)

3)      A heat-stable DNA polymerase.

 

From there the PCR reaction goes through 3 steps:

 

1)      Strand separation or denaturation. The two strands of the parent DNA molecule are separated by heating the solution to 95º C for 15s.

2)      Hybridization or annealing of two oligonucleotide primers (one is a reverse primer and the other is a forward primer). The solution is quickly cooled to 54ºC to let the primers anneal to a DNA strand. One primer anneals to the 3’ end of the target (template strand) while the other primer anneals to the 3’ end of the complementary target strand. Then each copy will be the template in the next cycle. This primer annealing also depends on the melting temp (Tm) of the primer.

3)      DNA synthesis or elongation or extension. The solution is then heated to 72ºC the optimal temperature for Taq DNA polymerase. This is a polymerase from a thermophilic bacterium, Thermus aquaticus, which lives in hot springs. This polymerase elongates both primers in the direction of the target sequence because DNA synthesis is in the 5’ to 3’ direction. DNA synthesis continues on both strands and continues beyond the target sequence (Berg et al.149-150).

 

All 3 steps above are considered as one cycle; it takes about 25-40 cycles in order to amplify DNA template. The number of cycles depends on the amount of DNA available and how much PCR product you want to yield. To carry out the PCR we will use a thermal cycler that is programmed with a protocol that goes through all three steps of a cycle, for a total of 35 cycles. See PCR animation.

 

 

Other reagents necessary for PCR

Remember we will be replicating DNA in vitro so we will need all the ingredients, which a cell uses in vivo, in the micro centrifuge tubes. The dNTPs are all four nucleotides, dATP, dGTP, dCTP, and dTTP; and need to be added in excess for the reaction to work. The two universal primers are also in excess, they are universal because they work with any Eubacteria species. The reaction is driven to completion while the amplification is in a 10mM Tris-HCl buffer. For the correct ionic strength 50mM of KCl must be in the buffer. The buffer and enzyme cofactor MgCl are necessary for the Taq DNA polymerase (pol) to work. In the reaction excess MgCl allows there to be free Magnesium ion (Mg 2+) to act as an enzyme cofactor. The primers, template, PCR product and dNTPs also bind Mg 2+. Mg 2+ concentration should always be greater than the total dNTP concentration (White). Of course water (H2O) must be present for the reaction to work, however, it must be special H2O that is free of DNA or RNA. Due PCR sensitivity you have to use this water. Most important you need DNA to run a PCR we will be using bacterial suspensions, made by you, for this lab. 

 

How many copies of the target does PCR give us?

The more times the three PCR cycles are repeated the more DNA you can obtain. This is because every cycle of a PCR reaction theoretically doubles the amount of target copies, so we expect a geometric amplification. In other words PCR is an exponential process.

One could use this formula to calculate the theoretical output of any input:

 

Y = X ( 1 + efficiency ) n                                                       Y = amount of amplification target

X = input copy number

n = number of cycles

efficiency factor is given for each cycle

 

In a theoretical world 25 cycles could give 33 million copies. However, the process is self-limiting and you usually obtain something between 10 5 and 10 9 fold (White).

Since PCR is so sensitive we should always use a positive and negative control. The positive control should be a PCR mix with DNA known to work in amplification. The negative control should be a PCR mix without DNA. The experimental part should be your PCR mix with DNA. 

 

Where is PCR used?

PCR is used in molecular biology and genetic disease research to identify new genes. It is useful when working with degraded DNA seen in fields such as anthropology and evolution. Due to the high sensitivity and particularity of PCR it has also been useful for human identification in war as well as in crime labs. Scientists have also used PCR in plant and animal breeding to look for specific traits. PCR has been used for quickly identifying environmental and food pathogens.  PCR has been successful in detecting viral diseases, and in speeding up gene discovery. Scientists have also been able to measure crossover frequency in a single sperm cell. Mixed samples can even be analyzed to use as forensic evidence (White). 

           

How do we see our results after running a PCR?

The most common way of seeing the results of a PCR is by running a gel electrophoresis.  The PCR product is pipetted into a special agar that will separate the DNA fragments according to their weight by using electricity. Remember, DNA has a sugar backbone that is negatively charged in a neutral to basic solution, the buffer solution, which is 1X TBE. After running the gel the fragments can be seen with a UV light. The reason we can see the fragments with UV light is due to the Ethidium bromide dye that has been added to the gel. This dye will adhere to the DNA in the gel and when the gel is finished it can be viewed under a UV light and can be photographed. Because Ethidium bromide is a known mutagen and carcinogenic the gel will be run UNDER THE HOOD AND GLOVES MUST BE WORN WHEN YOU GO TO THE HOOD. 

 

What will we do in lab with PCR?

Your TA or assistant lab coordinator will do one of the very first steps. All of the required PCR reagents need to be removed from the minus 20˚C freezer and placed in ice in order for the reagents to thaw this takes approximately 20 minutes. After the reagents thaw place them back in the ice. If reagents do not thaw quickly enough they may be thawed at room temperature so the lab can start. BUT DO NOT LEAVE REAGENTS OUT OF THE ICE FOR TOO LONG. The necessary reagents to thaw are:

 

            1 tube of Master Mix  

            1 tube of 1494 R primer

            1 tube of 530 F primer

            1 tube of DNA suspension used for positive control (TA will use this)

 

While the PCR reagents thaw each student can make their own DNA suspensions that will be used for the PCR reaction. A turbid bacterial broth or colonies from a plate may be used to create the suspension.

 

IMPORTANT REMINDERS

 

  • ALWAYS keep PCR reagents on ICE.
  • Remember the centrifuge tubes are sterile so as soon as you are ready to start making the cell suspensions get your tubes and close them so that nothing contaminates your centrifuge tubes.
  • Gloves are good to use to keep your tubes from getting contaminated.
  • While making the cell suspension you should NOT flame the centrifuge tubes they might melt.
  • Keep your tubes closed and make sure you do not cross contaminate with the tips of the micropipettes.
  • Micropipettes come in different sizes and so do the pipette tips make sure you are using the correct pipette and tip for the amount you need to pick up.
  • The p20 micro pipette is only accurate for picking up 2μL - 20μL
  • A p200 micro pipette is only accurate for picking up 20μL - 200μL
  • The p1000 micro pipette is only accurate for picking up 100μL - 1000μL
  • VERY IMPORTANT when using the micropipettes be careful and avoid contaminating the micropipettes by slowly releasing the plunger.
  • When using the micropipette you will need to stop pushing the plunger when you feel it stop the first time. The second stop will give you more than what you measured. 
  • Remember, if you are not sure about the last thing you did with your pipette tip then you should just get a clean one.
  • Dirty pipette tips should all be autoclaved so they are to be discarded in the tin cans with red bags.
  • Please balance the micro centrifuge machine by placing another tube across from your tube both tubes should have approximately the same amount of liquid, remember you are balancing.
  • You may use the balance tubes or another student’s micro centrifuge tube. 
  • Make sure you know where you place your tube or you will not know which tube is which. The best thing to do is to label the micro centrifuge tubes (LABEL EVERYTHING). Or else you will have to start over.
  • If you have never used a micropipette practice with autoclaved DI water

 

Part I:  Amplification (Day 1)

       

~Procedure~

 Cell suspension from a plate:

           

1.      Measure out 100μL of autoclaved DI water and add to an autoclaved micro centrifuge tube.

 

2.      Aseptically pick up a lot of unknown bacteria using a loop and shake the bacteria in the autoclaved DI H20 in your micro centrifuge tube close the tube. 

 

3.      Place your closed micro centrifuge tube in the vortex machine to mix well the bacterial cells.

 

4.      Cell suspension should be thick and uniform if you are unsure, ask or compare with TA’s cell suspension.

 

5.      Get a sterile PCR tube close it, LABEL it, and then open it to pipette 5μL of your (well mixed) cell suspension. Close and place PCR tubes in ice.

 

Cell suspension from a bacterial broth:

 

1.      Aseptically micropipette 100μL of a very turbid mixed bacterial broth, close the tube.

 

Follow steps 3-5 from up above.

 

PCR protocol

The next step is adding all the PCR reagents together into one sterile micro centrifuge tube, labeled Master Mix. Since the amounts necessary for a single 50μL PCR reaction are difficult to measure each lab will make 1 master mix. Below is the breakdown of what amount is measured into each micro centrifuge tube per number of reactions. 

 

# of reactions

25

Promega Master mix (μL)

625

1494 R primer (μL)

12.5

530 F primer (μL)

12.5

Nuclease free H20 (μL)

612

Total (μL)

 

 

The amount of reagent per 50μL reaction is as follows these numbers are shown so that you can see what your own reaction should have. Do not try to measure 0.26μL you won’t be able to see it and we don’t have a micropipette that measures that small amount.

 

# of reactions

1

Promega Master mix (μL)

25

1494 R primer (μL)

0.5

530 F primer (μL)

0.5

Nuclease free H20 (μL)

19

Total (μL)

45

 

If you add up the total for 1 reaction you’ll notice it is missing 5μL of the cell suspension that you will add to the PCR tube NOT the master mix.

 

Making the Master Mix (TA):

 

  1. Turn the dial of the micropipette so that you are measuring the proper amount of PCR reagent for your group. 

 

  1. All of the PCR reagents must be thoroughly mixed just prior to measuring - flicking the tube of the reagent you need to measure does this. Make sure all the liquid has uniform turbidity.

 

  1. DO NOT vortex the PCR reagents, this will destroy them.

 

  1. Put the proper micropipette tip on the micropipette.

 

  1. Push the plunger down to the first stop

 

  1. Be sure ONLY the pipette tip touches the inside of the tube and slowly release the plunger and be sure you picked up the liquid you are measuring. Avoid contamination.

 

  1. Put your liquid into the proper tube by pushing the plunger to ONLY the first stop. 

 

  1. PCR reagents go into the Master Mix micro centrifuge tube.

 

The Student:

 

  1. When done with the Master Mix, mix it well, and pipette 45μL of it and add it to your own PCR tube. Close your tube and place in ice.

 

  1. When the class is done with PCR tubes they must all be mixed by flicking just prior to loading into the thermal cycler when it has reached a temperature of 95 ºC

 

BE CAREFUL the cycler is HOT and will burn your skin if the plate is touched just insert the tubes and lock the cycler.

 

Part II: Running Gel Electrophoresis (Day 2)

 

~Overview of Electrophoresis~

As we said before to see your PCR results you need to run a gel. Due to the lengthy PCR process the gels will be run next lab meeting. DO NOT MISS any parts of the PCR lab; PCR lab products degrade quickly and make-ups are not possible.

 

Your TA or assistant lab coordinator will pour the electrophoresis gels because this is where the Ethidium bromide is located. Whenever you need to work with the gel; gloves MUST be worn to avoid contact with the Ethidium bromide dye, it is MUTAGENIC.

 

TA or assistant lab coordinator will take pictures of the gels because the use of a UV light is required. Remember UV is also mutagenic /carcinogenic so it must be used carefully and protection must be used as well.

 

PCR tubes with product have been stored at -20˚C; they need to be thawed in the fridge at 4˚C or in ice. This should be quick because you only have 50μL in these PCR tubes.

 

Your TA should also thaw the ladder, which is a DNA marker that will be used to show you where the 1500 Kb is located on the finished gel. The ladder is mixed with nuclease free H20 and some 6X dye. TAs will load the ladder and the positive and negative controls.

 

The electrophoresis gel is in a buffer solution that ensures the DNA will be negatively charged. The gel also has 32 wells in it that are formed with combs inserted into the gel before it has hardened. You will pipette your PCR product + dye into these wells. When doing this you need to be careful not to break the gel, which has a consistency of gelatin. To avoid breaking the gel, steady your hand and place the pipette tip in the buffer solution just above the well you chose. DO NOT TOUCH the gel you don’t need to touch it with the pipette tip. Just slowly push the plunger to the first stop and your solution will fall right into the well. If this is done successful you have loaded a well.

If you break a well let your TA know.

 

The reason the product will fall is due to the dye you added to the PCR product the dye makes the product heavy. This dye is also used so you are able to see what wells have product and when the gel needs to be stopped. If a gel is never stopped the product runs right off the gel and into the buffer. To stop a gel all you need to do is turn off the electricity.

  

~Procedure~ 

 

 

                  1.  Take 12 μL of your thawed PCR product in a p20 micropippete.

 

                  2.  Bring micropippete to gel, and carefully place tip near one unused well for

                         dispensing.

 

                  3.  Release product slowly into gel, allowing it to fall into the lower well.

 

                   4.  The green dye has a reagent that allows the product to be heavier than the buffer

                         it is in therefore allowing it to fall into well.

  

Once everyone has put their PCR products into the wells the electrodes need to be checked and the machine needs to be turned on at 150V because it is such a large gel.

 

Why check the electrodes? Because you want your gel to run from where you loaded the wells to the bottom of the gel, if it goes the wrong way you will run your product into the buffer. You need the negative electrode on the loaded wells side and a positive electrode on side without wells; that way your product, which is negatively, charged moves towards the positive end. Running a gel takes about 40 – 60 minutes.

 

When the gel is done a picture will be taken and you can check to see if you properly ran a PCR and gel and see the proof of the 16S gene amplified at 1500 bp. 

 

Gel Outcomes: 

 

* A positive result for bacteria will have the 16S Gene deposited at 1500 bp, such as the cartoon rendition in Well 2.

 

Acknowledgements

 

I would like to thank David Chang, Shyama Malwane, Linh Nguyen, and Dr. Eduardo Robleto for their expertise and support.

 

References

 

White, Bruce A., ed. PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering. Methods in Molecular Biology, Volume 67. Totowa: Humana Press Inc 1997.

Berg, J.M., Stryer, L., and Tymoczko, J.L. Biochemistry 5th ed. New York: W. H. Freeman and Company, 2002. 

 

Revised by N. Fester on November 7, 2005