LAB: Molecular Techniques

Overview
Special enzymes termed restriction enzymes have been discovered in many different bacteria and other single-
celled organisms. These restriction enzymes are able to scan along a length of DNA looking for a particular
sequence of bases that they recognize. This recognition site or sequence is generally from 4 to 6 base pairs in
length. Once it is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix.
The restriction enzyme will continue to do this along the full length of the DNA molecule which will then break
into fragments. The size of these fragments is measured in base pairs or kilobase (1000 bases) pairs.

Most restriction endonucleases recognize palindromic or partially palindromic sites. A palindrome is defined as
dyad symmetry around an axis. For example, EcoRI:

Since the recognition site or sequence of base pairs is known for each restriction enzyme, we can use this to
form a detailed analysis of the sequence of bases in specific regions of the DNA in which we are interested.

Purpose
 Understand how to use a restriction digestion analysis of a sample of DNA.
 Learn to separate DNA on an agarose gel using electrophoresis.

Materials
1. Microcentrifuge tubes
2. Microtube rack
3. Micropipette and sterile tips
4. Ice bucket with crushed ice for the following
5. gDNA (from DNA extraction lab)
6. Restriction enzymes
7. Distilled water
8. 250 ml beaker
9. Electrophoresis chamber
10. Agarose gel (0.8%)
11. Loading dye
12. DNA ladder/marker
13. Power supply
14. Loading dye
15. TBE solution (1X)
Lab Protocol

Part 1. Restriction enzyme digestion of DNA

TIPS BEFORE STARTING:

As you add each reagent to a 1.5 ml microfuge tube, stir it in well with the pipet tip and always change your tip
in between steps - NEVER double dip your tip!
*VERY IMPORTANT! * Do not dispense the liquids onto the sides of the tube. Always dispense the volumes
into the very bottom of the tube!
Enzymes are stored in glycerol and will never freeze and are ready to be used immediately. They never should
be out of the table freezer block or an ice bucket. However, the buffer must be completely thawed before use.
(Gently flick the tube and spin the contents to the bottom).
Keep your reactions, buffers and enzymes at 4°C on ice until you are ready to incubate your samples.

Procedure: (Take your time! Make sure you’re doing each step correctly!)

 Combine the following in a microfuge tube in order

Double digest Prep: Single digest Prep:

1µl 10x Buffer 1µl 10x Buffer
6.0µl H2O 6.5µl H2O
2µl DNA 2µl DNA
0.5µl Enzyme 1 0.5µl Enzyme 1
0.5µl Enzyme 2

Enzyme: 0.5µl of enzyme is plenty for a miniprep digestion. Do not use more enzyme than 10% of the final
reaction volume (i.e. not more than 1µl in this case). This is because the enzyme storage buffer contains
antifreeze (glycerol) to allow it to survive at –20°C. The glycerol will inhibit the digestion if present in
sufficient quantities. You may be digesting your DNA with two (or more) enzymes. Rule of thumb: popular
old-fashioned enzymes (EcoRI, BamHI) are the best and most forgiving,

 Mix well by pipetting slowly up and down approximately 5x. Be gentle, and do not vortex.

 Spin the samples for 5 seconds in a balanced microcentrifuge, or flick them to collect the mixture at the
bottom of the tube.

 Incubate for 1 hour at 37°C in a water bath.

 After digestion, incubate your samples for 80°C for 20 minutes (in heat block). It is important you heat
inactivate your samples after digestion.

 Place the samples on ice and store for analysis

Part 2. Agarose gel electrophoresis
Agarose gel electrophoresis is the easiest and common way of separating and analyzing DNA. The purpose of
the gel might be to look at the DNA, to quantify it or to isolate a particular band. The DNA is visualized in the
gel by addition of DNA-binding dyes that are fluorescent, meaning that they absorb invisible UV light and
transmit the energy as visible light.

What percentage gel?

Most agarose gels are made between 0.7% and 2%. A 0.7% gel will show good separation (resolution) of large
DNA fragments (5–10 kb) and a 2% gel will show good resolution for small fragments (0.2–1 kb). Some people
go as high as 3% for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this
case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are
often brittle and do not set evenly. In this lab we will use 0.8% agarose gel (pre-made).

Preparing the samples

 Add an appropriate amount of loading buffer into each tube and leave the tip in the tube. The tip will be
used again to load the gel. The loading buffer gives color and density to the sample to make it easy to
load into the wells. Also, the dyes are negatively charged in neutral buffers and thus move in the same
direction as the DNA during electrophoresis.

 Place your gel in the chamber and cover the gel with TBE buffer.

 Load 2 µL of DNA ladder (ready-mixed with loading buffer) to the first well

It is best to not use the outer wells because they are the most likely to run aberrantly.

 Continue loading the samples and finish with a final lane of marker. Label and record the content of
each well/lane as you are loading your samples.
DNA will migrate away from the wells. DNA is negatively charged, therefore, when an electric current is
applied to the gel, DNA will migrate towards the positively charged electrode.

 Close the gel tank, switch on the power-source and run the gel

For example, if the electrodes are 10 cm apart then run the gel at 50 V. It is fine to run the gel slower than this
but do not run any faster. Above 5 V/cm the agarose may heat up and begin to melt with disastrous effects on
your gel's resolution. Some people run the gel slowly at first (e.g. 2 V/cm for 10 minutes) to allow the DNA to
move into the gel slowly and evenly, and then speed up the gel later. This may give better resolution.

 Check that a current is flowing

You can check this on the power-source, the milliamps should be in the same ball-park as the voltage, but the
best way is to look at the electrodes and check that they are evolving gas (i.e. bubbles). If not then check the
connections, that the power-source is plugged in, etc.

 Monitor the progress of the gel by reference to the marker dye.

 Stop the gel when the marker dye has run 3/4 the length of the gel.

 Switch off and unplug the gel tank and carry the gel to the UV light-box.

 Carefully place the gel onto the glass surface of the light-box. UV is carcinogenic and must not be
allowed to shine on naked skin or eyes. So, wear face protection, gloves and long sleeves.

 Take a picture of your gel for analysis.