Purification of a Gene

(BE-306)
MATERIALS INCLUDED WITH THE KIT
This kit has enough materials and reagents for 24 students (six groups of four students).
Checklist
‰ 1 vial DNA: Plasmid 1 (digested)
‰ 1 vial DNA: Plasmid 2 (digested)
‰ 1 pack Agarose
‰ 1 vial DNA Stain (500X)
‰ 1 vial DNA Ladder (1kb)
‰ 1 vial DNA Loading Buffer (6X)
‰ 1 bottle TAE Buffer (50X)
‰ 1 bottle Gel Dissolving Buffer
‰ 1 bottle Resin Wash Buffer 1
‰ 1 bottle Resin Wash Buffer 2
‰ 1 vial DNA Binding Resin
‰ 1 vial TE Buffer
‰ 60 Centrifuge Tubes (1.5ml)
‰ 6 Gel Picks
‰ 6 Wide Bore Pipette Tips

SPECIAL HANDLING INSTRUCTIONS

¾ Store Plasmids, DNA ladder (1kb) and DNA Loading Buffer (6X) frozen at -20°C.
¾ All other reagents can be stored at room temperature.

The majority of reagents and components supplied in the BioScience Excellence™ kits are non
toxic and are safe to handle, however good laboratory procedures should be used at all times. This
includes wearing lab coats, gloves and safety goggles.
For further details on reagents please review the Material Safety Data Sheets (MSDS).
The following items need to be used with particular caution.

Part # Name Hazard

R141 Resin Wash Buffer 2 Flammable
D161 DNA Stain (500X) Flammable

ADDITIONAL EQUIPMENT REQUIRED

¾ Agarose Electrophoresis Equipment
¾ Waterbath or beaker and thermometer
¾ Balance (Optional)
¾ Low speed centrifuge for 1.5-2ml tubes

TIME REQUIRED
¾ Day 1: 3 hours

Bioscience Excellence™ Teacher’s Guidebook 1

Rev:070708
 Purification of a Gene
(BE-306)

OBJECTIVES
¾ Separate DNA fragments by agarose electrophoresis.
¾ Purify a gene of interest from the agarose gel.

BACKGROUND
Agarose gel electrophoresis is routinely used as an analytical tool for the examination of DNA fragments, however
an equally important use is in the separation and purification of DNA fragments.

Several methods exist for the purification of DNA fragments from agarose. The choice of technique is a matter of
preference and cost, but all techniques begin with the excision of the DNA band of interest from an agarose gel.

The first and most commonly used approach is the melting of the agarose and the binding of the released DNA to a
solid support, such as glass fibers or silica. At high salt concentration and neutral or low pH, DNA molecules have a high
binding affinity for these supports, allowing for the easy capture and subsequent elution of the DNA. A common problem
utilizing glass fibers for the purification of DNA is the low visibility of the resins in solution. This kit contains a special pink
resin that allows for high visibility and greater ease of use.

A second method for the purification of DNA from agarose is electroelution, which uses a current to migrate the
DNA out of the agarose. Originally, the excised piece of agarose is normally placed in to a small piece of dialysis tubing, an
electric current is passed across the dialysis tubing that causes the DNA to migrate out of the piece of agarose. The purified
DNA can then be removed from the dialysis tubing. Today several specialized apparatus based on this technique are
routinely available, which allow for easier excision and electroelution of the DNA. G-Biosciences manufactures G-
™
CAPSULE , an electroelution device for rapid purification of proteins and nucleic acids from electrophoresis gels. Figure 1
™
depicts a schematic of how the G-CAPSULE works.

™
Figure 1: G-Biosciences electroelution device, G-CAPSULE
Another method uses an enzyme to digest the agarose. The agarose, containing the DNA, is excised from the gel
and then melted in an appropriate buffer. Once the gel piece has melted a β-agarose digesting enzyme is added. The enzyme
digests the carbohydrate backbone of the agarose releasing small soluble oligosaccharides. The DNA can be used at this
stage, however an ethanol precipitation is normally utilized after the enzyme incubation time, to ensure contaminant free
purified DNA.

Ion exchange chromatography is another routinely used technique for the purification of nucleic acids. Basically the
negative charged properties of the phosphate backbone of the nucleic acids is bound to an anionic resin, such as DEAE
(diethylaminoethyl) -cellulose membrane. Impurities that fail to bind to the membrane are washed away and the nucleic
acids are eluted with high salt buffers. There are numerous commercially available products based on this method. The
DEAE-cellulose is incorporated into a small spin column. The DNA is excised in the agarose, melted and then applied to the
column for binding. The use of spin columns are more user friendly.

This kit can be linked to the polymerase chain reaction kit, for the purification of the PCR products, or the DNA
restriction digestion analysis, for the purification of digested fragments.

Bioscience Excellence™ Teacher’s Guidebook 2

 Purification of a Gene
(BE-306)
TEACHER’S PRE EXPERIMENT SET UP
Agarose Gel
1. In order to visualize and separate the DNA fragments an agarose gel will be required. The number of wells required
depends on how this kit is used.
a. If used as a stand alone kit 2 wells are required per group.
b. If used in conjunction with the Polymerase Chain Reaction, each student requires 1 sample well and
additional sample wells are required for reference markers.
c. For use with the DNA Restriction Digestion kit, each student requires 2 sample wells and additional sample
wells are required for reference markers.

2. You may use your own equipment and supplies or use G-Biosciences’ “Introduction to Agarose Electrophoresis” kit
(Cat. # BE-304). A 1% agarose gel is recommended.

3. Following the purification of the DNA, a sample of the purified DNA can be visualized on an agarose gel. Prepare
an agarose gel to visualize a select number of purified DNA.

Preparation of agarose gel

Make 1-2 hours before the experiment.
Wear heat protective gloves throughout the agarose melting and pouring procedure

1. Prepare running buffer: In a clean two liter container, add the entire contents of the 50X running buffer and add two
liters of ultra pure water to make a 1X running buffer solution. Stir until thoroughly mixed.

2. Prepare agarose: In a clean, glass 1000ml container add the entire contents of the agarose pack and add 500ml of the
running buffer from step 1.

3. Heat the solution in a microwave on full power, using 10 second bursts, or use a boiling waterbath. Check to see if
all the agarose has dissolved. Continue until agarose has dissolved.
DO NOT BOIL. The agarose gets very hot, very quickly and can cause severe burns. Wear heat protective
gloves throughout the melting and pouring procedure

4. Once the agarose has cooled to the point it can be held comfortably in your hand, pour the agarose into the gel
casting mould as per the manufacturer’s instructions.

5. Once the gels have set, remove the comb, transfer to the running apparatus and cover with the running buffer until
ready to use.

Prepare Dilute DNA Staining Solution

The DNA stain solution is a safe alternative to the carcinogenic ethidium bromide routinely used in laboratories. Ethidium
bromide is more sensitive, but is a strong carcinogen and requires exposure to UV radiation. The DNA stain contains
methylene blue.

1. Add 100ml 1X TAE buffer to a 1 liter container and add 900ml deionized water to make a 0.1X TAE buffer. Stir
until thoroughly mixed. The 0.1X TAE buffer is used to dilute the DNA Stain and also as the destaining solution.

2. To 500ml 0.1X TAE buffer add 1ml 500X DNA Stain. Stir until thoroughly mixed

Prepare the reference markers

1. Add 25µl ultra pure water to the lyophilized 1kb DNA ladder, dissolve by gently pipetting up and down 5-6 times.

Bioscience Excellence™ Teacher’s Guidebook 3

 Purification of a Gene
(BE-306)
2. Add 5µl 6X Loading Buffer, mix by gently pipetting up and down 5-6 times.

3. Load 10µl into each reference well.

Reconstitute DNA
1. Add 150µl ultra pure water to each tube of DNA sample, dissolve by gently pipetting up and down 5-6 times.

2. OPTIONAL: Aliquot the 25µl DNA vector into clean tubes and give one tube of each DNA vector to each group of
students

Prepare Resin Wash Buffer

1. Prior to use, add the entire contents of the Resin Wash Buffer 2 bottle to the Resin Wash Buffer 1 bottle. Shake
vigorously to mix.

2. Store at -20°C.

Prepare TE Buffer
1. The TE buffer should be placed in at 55-65°C waterbath prior to commencement of the experiment.

MATERIALS FOR EACH GROUP

Supply each group with the following components. Several components are shared by the whole class and should be kept on
a communal table.
DNA Sample(s)
1 vial DNA Loading buffer
1 bottle Gel Dissolving Buffer (shared with class)
1 bottle Resin Wash Buffer (shared with class)
1 vial DNA Binding Resin (shared with class)
1 vial TE Buffer (shared with class)
8 centrifuge tubes (1.5ml)
1 Gel pick

PROCEDURE
1. Label a tube for each DNA band to be excised with your name and an abbreviated name of the DNA fragment to be
isolated.

2. Add 6X DNA loading buffer to your DNA samples to give a final 1X concentration. For example, to the
supplied 25µl Plasmid 1 (digested) and Plasmid 2 (digested) add 5µl 6X loading buffer.

3. Load 30µl onto a 1% agarose gel as instructed by your supervisor. Your teacher/ supervisor will load a reference
lane to determine the size of the DNA (see image for reference markers).

4. Once the samples are all loaded apply a current and migrate at 12-15V/cm. For an 8cm long gel run at 96-120
volts.

5. Once the blue dye front has migrated ¾ the length of the gel, turn off the power and carefully transfer the gel to a
staining tray.

CAUTION: Agarose gels are very fragile, handle with extreme care.

Bioscience Excellence™ Teacher’s Guidebook 4

 Purification of a Gene
(BE-306)
6. Add sufficient diluted DNA Stain to cover the gels and place on a slow (less than 60rpm) shaker for 1-4 hours at room
temperature or overnight at 4°C.
If shaker is too fast the gels will break. As an alternative, leave the gel at room temperature for 2-4 hours or
overnight at 4°C without shaking.

7. If DNA bands are hard to see after staining, due to a high background, then destain with 0.1X running buffer (0.1X
TAE) for 30-60 minutes. To help visualize bands, place gel on a sheet of white paper.

8. Taking it in turns, each student uses the gel pick to carefully excise a DNA band. In the Plasmid 1 (digested) sample,
bands at 2.2 and 0.8kb can be excised and bands of 1.6 and 1.1kbp can by excised in the Plasmid 2 (digested) lane.

9. Using the cutting edge of the gel pick push into the agarose gel until it hits the gel tray (Figure 2 (1)). Remove by tilting
and pulling up (Figure 2 (2&3))

Figure 2: How to use gel pick.

10. Using a pipette tip push the excised piece of agarose into a labeled 1.5ml centrifuge tube.

11. Zero a balance with an empty tube and weigh and record the weight of the tubes containing the agarose.
Weight:

12. Add approximately 3 volumes of Gel Dissolving Buffer to the tube with the agarose.
This is normally about 300µl. In the absence of a balance and to save time, instruct the students to add
300µl of Gel Dissolving Buffer.

13. Place the tube in a 55-65°C waterbath for 5-10 minutes, or until the agarose has completely melted.

14. Place the tubes on ice to cool for 2 minutes.

15. In the meantime, vigorously flick, shake or vortex the DNA Binding Resin to thoroughly resuspend the glass fiber.

16. Add 20µl DNA Binding Resin to the tube using the Wide Bore Pipette tip and return the tube to the ice bucket for 5
minutes. Shake the tube every 0.5-1 minute to resuspend the resin and increase its binding efficiency.

17. Centrifuge the tubes for 30 seconds to pellet the resin.

18. Remove the supernatant and discard.

19. Add 0.25ml Gel Dissolving Buffer, resuspend the resin by gently flicking the tube and repeat steps 17 and 18.

Bioscience Excellence™ Teacher’s Guidebook 5

 Purification of a Gene
(BE-306)
20. Add 0.5ml Resin Wash buffer, resuspend the resin by gently flicking and repeat steps 17 and 18 again.

21. Wash the resin a second time by repeating step 20.

22. After the final wash, centrifuge the tube for 30 seconds and remove any remaining supernatant with a pipette. The
Resin Wash Buffer contains ethanol that can inhibit DNA elution and any subsequent experiments using your eluted
DNA.

23. Leave the tube open at room temperature for 10-30 minutes to allow any residual ethanol to evaporate.

24. Once all the residual DNA Wash Buffer has evaporated, add 30µl prewarmed TE buffer and gently flick the tube to
resuspend the resin. Incubate at room temperature for 5 minutes to elute the DNA.

25. Centrifuge the tubes for 30 seconds and remove the supernatant to a clean labeled tube. This is your purified DNA that
can be visualized on a DNA gel or used in other molecular biology procedures, for example DNA ligation.
OPTIONAL: A small sample of the purified DNA can be used in a DNA assay to measure the concentration
of the DNA or it can be run on an agarose gel to visualize the purified DNA.

RESULTS, ANALYSIS & ASSESSMENT

The results and analysis is dependent on how the purified DNA is used. Design your own questions for the
students depending on how the purified DNA was assayed and used.

Bioscience Excellence™ Teacher’s Guidebook 6