Restriction digest

Our first procedure will be to prepare restriction digests of our DNA samples. Each person will receive a sample of the plasmid vector containing our gene insert. Make sure that you write down the identity of the sample.

Restriction digests require very small amounts of reagents to be added. Since this exercise is a check of our samples and we do not want to use all of them up, we will work with the barest minimum volumes.

The reagents required for our digest are :

  • DNA Sample – generally added in the same proportion as the restriction enzyme
  • Restriction Enzyme – generally added at 10% of the final volume
  • 10X Buffer – a solution of salts which maintain the correct pH for the enzyme to function. “10X” refers to the fact that it is ten times more concentrated than it needs to be and so must be diluted by the addition of the other reagents. The volume of buffer used must be 10% of the final volume. There may be different buffers required for each restriction enzyme
  • BSA – “Bovine Serum Albumin” a solution of protein derived from the blood of cows. This reagent stabilizes the enzyme reaction mixture, although it is not needed for all restriction enzymes
  • Water – used to make up the reaction mixture to the final volume

We will be performing a SalI restriction digest on our sample to check the orientation of our inserts.

The following table indicates the volumes needed for our restriction digest :

Tube Sample
10X Buffer
10X BSA Water Total
Sample 1µL 1µL 1µL 1µL 6µL 10µL
  • Prepare a tube containing the reagents listed in the table above.
  • Incubate the tubes at 37°C for 1-2 hours

Agarose gel electrophoresis

In order to check whether the orientation of our insert is correct, we need to examine the size of the DNA fragments which result from our restriction digest. 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 - a 1 in 50 dilution. The following calculations are used to find out the volumes needed:

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

Preparing the gel 

The gel we will be using is 0.8% agarose. This means that we should add 0.8g of agarose to every 100mL buffer. We need more than this amount as each gel needs 40mL and we will be doing 4 gels, so 4 x 40 = 160mL). The new mass needed is therefore 160mL / 100mL x 0.8 = 1.28g

  • Suspend 1.28g of agarose in 160mL of water.
  • Microwave the solution on HIGH for 2 minutes (for a small gel) in 30 second bursts. Make sure that the agarose is completely dissolved by swirling the heated mixture. Allow it to cool for 3 minutes.

Take care - do not put the lid on the flask while microwaving

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 16µL 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 trays.  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 going to use all 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 digest 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).make sure you record which lane your sample is in

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 - electrophoresis involves high voltages

  • Switch off the power pack and take the gel to the transilluminator. Take a photograph, print off and glue into your workbook. Annotate the photograph, 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.

Map of 1kb DNA 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 in the figure to the right.

In order to visualize the DNA bands, we need to place the gel on a UV Transilluminator. The SYBR-Safe dye we added to our gel binds to the DNA and fluoresces green under ultraviolet light.

  • Place your gel on the transilluminator, close the plastic shield and switch it on
  • Observe the location of DNA bands in the lane that you loaded.
  • You should either see a single band at 3.7kb (incorrect orientation) or bands at 3kb and 0.7kb (correct orientation). Was the insert in your sample oriented correctly or incorrectly ?

Your tutors will take your gel to a transilluminator with a camera attached to photograph it. The image will be forwarded to your teacher for a permanent record of the experiment (Note that the photograph will be in grayscale, as colour cameras are not sensitive enough to detect the signal from the SYBR-Safe).