Polymerase Chain Reaction

The first step is a simple one – we need to set up our PCR tubes. Any PCR reaction uses the same basic ingredients :

  • Deoxyribonucleoside triphosphates (dNTPs) – adenosine triphosphate, thymidine triphosphate, cytidine triphosphate and guanosine triphosphate – these are the raw materials for making our DNA during PCR.
  • polymerase enzyme to make the DNA (in this case Pfu Polymerase)
  • Buffer – this is a solution of salts and ions which stabilises the reaction by ensuring that the pH remains constant.
  • Primers (forward and reverse).
  • Plasmid vector containing the PLK1 gene to act as our template.
  • Nuclease-free distilled water to make the volume up to 50µL.

Keep in mind that PCR is a notoriously sensitive procedure – it will amplify any DNA it encounters in the reaction mixture, so long as the primers bind.  Therefore, it is important to limit contamination in this part of the experiment.  We use specially purified water and sterile tips on our micropipettes.

Many of the volumes are quite small and will need the use of the P2 micropipette.  To ensure that the majority of the material is dispensed, add the largest volume (the dH2O) first and draw the liquid up and down inside the pipette tip a few times.  Make sure you dispose of your tips between samples.

You will be making up two tubes: a sample (which has all of the ingredients you need to make your gene fragment) and a negative control (which has everything except the DNA vector).  The inclusion of the control is a way of testing whether the reaction worked or not.

The volumes needed for the PCR reaction are provided below:

Tube
Label
dH2O 10X
Buffer
DNA
Template
dNTPs Forward
Primer
Reverse
Primer
Pfu II Ultra
Polymerase
Control 35.2µL 5µL 4.8µL 2µL 1µL 1µL 1µL
Sample 40µL 5µL - 2µL 1µL 1µL 1µL

 The thermal cycler in the SPARQed laboratory has been programmed for this experiment and the reaction will run for nearly 2½ hours. For your own information, the details of the program are:

95°C 30 seconds  
95°C 30 seconds  
60°C 30 seconds 30 Cycles
72°C 2 minutes  
72°C 10 minutes  
4°C Until Ready  
  • Place the tubes containing your test and control solutions into the thermal cycler, close the lid and run the program “pbx001”

Agarose gel electrophoresis

In order to check whether our PCR has been a success, we need to check the size of any fragments of DNA found in our test solution (our control solution, lacking template DNA, should contain no detectable DNA). We do this using agarose gel electrophoresis.

Preparation of TAE buffer

Electrophoresis uses an electric field to “push” DNA fragments through the gel. To ensure that this occurs efficiently, all of the DNA must have a negative charge (to ensure that it is pushed away from the negative terminal). This is done using a buffer which keeps the experiment at a pH where all of the DNA is negatively charged.

The buffer most commonly used in DNA analysis is TAE, which stands for Tris – Acetate – EDTA (where EDTA stands for ethylenediaminetetracetic acid). The buffer is usually made up at 50X concentration and then must be diluted when needed (this allows us to make and store large amounts of the buffer without having to remake it as often).

For the whole group, we will only need around 500mL of TAE buffer. You will need to prepare 500mL of 1X TAE buffer from the 50X stock solution provided. Perform the following calculations :

  • Total volume     = 500mL
  • 1/50 of 500mL     = 500 ÷ 50  =  ____ mL
  • Volume of 50X stock needed is ____ mL
  • Volume dH2O needed      = Total volume  - Volume stock needed

= 500mL – ____ mL

= ____ ml

  • Dilute ____ mL stock in ____ mL of dH2O
  • Use the calculations above to prepare 500mL of 1X TAE buffer

Preparation of gel

The gel used to studying DNA is made from agarose, a jelly-like substance derived from seaweed. This material is supplied in powder form, and must be dissolved in the TAE buffer. For our experiment, we require a gel containing 0.8% agarose, ie. 0.8g of agarose powder dissolved in 100mL of  buffer.

  • Weigh out 0.8g of agarose powder and suspend in 100mL of TAE buffer in a conical flask. One quantity is sufficient for the entire class
  • Microwave the solution on HIGH for 2 minutes (for a small gel).  Make sure that the agarose is completely dissolved by swirling the heated mixture roughly every 30 seconds.  Allow it to cool for 3 minutes.

Take care while heating agarose

Take care with hot agarose

  • Wipe a plastic gel tray and comb with 70% ethanol and place in the electrophoresis tank so that the rubber tubing forms a seal with the sides of the tank.
  • Add 10µL of SYBR-Safe into the melted agarose and swirl to mix. This substance is a dye which binds to the DNA and glows green under ultraviolet light – it allows us to see where the DNA has migrated in the gel.
  • Pour the melted agarose into the gel tray.  Place the comb into the right position and allow it to set for approximately one hour (this can be done faster by placing the gel tray in the refrigerator.
  • Carefully remove the comb from the gel.  Rotate the gel tray so that the wells are toward the negative (black) terminals (the top of the tank, assuming that the electrodes are on the right hand side).  Cover the gel with 1X TAE running buffer.

Loading the gel

The samples must now be loaded into the wells in the gel left by the comb. To make this process easier, we mix the samples with a blue dye and glycerol. The dye migrates before all of the DNA and we can use this to tell when to stop running the gel. The glycerol increases the density of the sample so that it sinks to the bottom of the well on loading. The dye is provided at 6X the required concentration. This means that we have to add it to the sample in a proportion which dilutes it 1 in 6 (ie. five times as much sample as dye). Use the following calculation to find out how much dye is needed to add to a given volume of sample :

  • We are only going to use 10µL of our digest product
  • If the volume of dye added is “x” :

x  +  Volume of DNA  =  6x

  • Volume of dye needed to add to 10µL of PCR product =  _______  µL
  • Prepare loading solutions for each of your samples and DNA ladder.
  • Load all of the loading solutions into separate wells in the gel (loading the DNA ladder last into a separate well on the left or right hand side of your gel).  Record where you have loaded each sample.

Running the gel

  • Run the gel at 80V.  There must be small bubbles rising from both ends of the electrophoresis chamber.  Check after 5 minutes to make sure the gel is running (i.e. the dye front has moved, is relatively straight and has run the correct direction).  Then allow the gel to run for the necessary amount of time (about 1 hour however, check that the dye front has almost run through the gel).

Take care with high voltage

  • Switch off the power pack and take the gel to the illuminator.  Take a photograph, print off and glue into your workbook in the space below.  Annotate the photograph using the ID table you completed above, indicating bands of interest.
  • Pour away the buffer from the electrophoresis tank and rinse well with water.  Rinse the gel tray and comb as well.

1kb ladderInterpreting your gel

 

Whenever we run a gel, we should always include a DNA “Ladder” which features fragments of DNA of known size. This ladder serves as a reference point to indicate the size of the DNA fragments in our sample. A map of the ladder we are using in this exercise is provided to the right.

Examine the photograph of your gel and check the sizes of bands. The polobox domain portion of the PLK1 gene is approximately 800 base pairs (or 0.8 kilobases) long. If the PCR has been a success, you should see a single band just below the “1.0kb” marker.