Genetic Engineering:
Directions for Laboratory Period 2

This Week's Objectives | This Week's Schedule | Materials | Procedure | Genetic Engineering Table of Contents

This Week's Objectives

Previously we cut two existing recombinant plasmids with restriction enzymes to generate DNA fragments. This week we will isolate and join together selected DNA fragments.

Specifically, we will...

• Isolate the desired restriction fragment from each plasmid.
• Join the two desired fragments together.
• Introduce (transform) the ligation mixture into host cells.

This Week's Schedule

1. Cast a preparative agarose gel (~1/2 hour).
2. Perform gel electrophoresis to isolate the desired DNA restriction fragments (~1 hour).
3. Excise the desired DNA fragments from the gel (~1/2 hour).
4. Set up a ligation reaction to join the two desired restriction fragments together (~40 minutes).
5. Transform the ligated DNA mixture into competent E. coli cells (next day; ~2 hours).

Materials

The following material is in each ice bucket:

• Restriction digested DNA from last week
• 6X loading dye
• 2X buffer + DNA ligase

In addition, the following equipment and supplies will also be available:

• Gel casting tray assembly (glass plate, plexiglass tray, comb, and tape)
• P-20 pipetman
• P-200 pipetman
• Sterile yellow pipet tips
• Sterile 1.5 ml tubes
• Microcentrifuge
• 16°C water bath
• 37°C water bath
• 42°C water bath
• 70°C water bath
• Pasteur pipets
• Gel boxes with lids
• 1x TAE Gel electrophoresis buffer
• Electrical power supplies
• Gloves
• Bunsen burners
• Weighing balance
• Low-melting temperature agarose
• Deionized H2O
• Competent E. coli cells
• SOC medium
• SOB + ampicillin agar plates
• Razor blades
• Bent glass rods
• 37°C incubator
• Ethidium bromide staining solution (@ 0.5 µg/ml)
• Ultraviolet transilluminator
• Ultraviolet-shielding safety goggles
• Film
• Film holder and hood
• Waste containers for solid and liquid materials contaminated with ethidium bromide

Procedure (To be performed with a partner)

Cast a preparative agarose gel

1. Assemble the gel-casting tray as follows: Place the glass plate in the tray, insert the comb into the appropriate notches, and seal the ends with tape. Place the assembled gel-casting tray out of the way on the lab bench so that the agarose poured in the next step can set undisturbed.
2. Dispense 25 ml of 1X TAE into a 100 ml flask. Add 0.25 g of low-melting temperature (LMT) agarose. Cover the flask loosely and microwave just to boiling. Using gloves, swirl to mix. Repeat until agarose is completely dissolved.
   Unlike regular agarose, LMT agarose dissolves at about 65°C (vs. ~90°C regularly) and gels just below 30°C (vs. ~40°C regularly). Because of the lower melting temperature, gel slices containing DNA fragments can be melted without denaturing (melting) the double-stranded DNA.

   LMT gels have less strength than regular gels; so use extra care when handling!

3. Once the solution has cooled to about 65°C, carefully pour the molten LMT agarose into the assembled gel-casting tray until full. Use a pipet tip to move large bubbles or solid debris to the sides, while the gel is still liquid. The gel will become cloudy as it solidifies (about 10 minutes). Be careful not to move or jar the casting tray while the agarose is solidifying. Touch a corner away from the comb to test whether the gel has solidified.
4. When solid, barely flood the gel surface with a small volume of gel electrophoresis buffer. Gently remove the comb, ensuring that the wells are not damaged. Remove the tape and place the tray in the gel box so that the wells are nearer the negative pole (the black electrode).
5. Fill the box with gel electrophoresis buffer to a level that just covers the entire surface of the gel.

 

Perform gel electrophoresis to isolate the desired DNA restriction fragments

1. Add 7 µl of the 6X loading dyes to the remaining 36 µl of each restriction-digested DNA (tubes labelled V and C from last week).
2. Load 20 µl of each sample into separate wells of the LMT gel.
   It's good laboratory practice to load only half of your sample, so that you have a reserve in case something happens. This way if the gel electrophoresis should fail, you won't have to go back to the beginning.
3. Run the gel at 100 V for about 60 minutes. This should be sufficient to isolate each desired fragment.
4. Transfer the gel assembly to the small staining container and add 0.5 µg/ml ethidium bromide solution until the gel is covered. Incubate 10 minutes at room temperature.

 

Excise the desired DNA fragments from the gel

1. Label and pre-weigh two sterile 1.5 ml tubes.
2. Visualize the stained DNA using UV light. Using a razor blade, excise a small gel slice containing each desired DNA fragment. Trim the gel slice to a minimal volume while minimizing exposure to UV light.
3. Transfer each small gel slice to a pre-weighed tube. Determine the weight of each gel slice.
4. Assuming that the density of the gel is 1 g / ml, calculate the volume of the gel slice. Then determine the concentration of the DNA insert, using the following equation:
   Concentration of insert = total amount of DNA loaded x (insert length / total plasmid length) ÷ volume of the gel slice

 

Set up the ligation reactions

1. Calculate the amount of insert and vector required for the ligation reaction according to the following rules:
   • Include equal (mass) amounts of each restriction fragment.
   • The combined volume must equal 10 µl.
2. Melt both gel slices by heating the tubes in a 70°C water bath for 10 - 15 minutes. Meanwhile, prewarm a sterile, labeled 1.5 ml tubes at 37°C.
3. To the prewarmed tube, add the volume of both fragments calculated above (step D.1). Mix gently by flicking the tube with your finger.
4. Incubate the tube at 37°C for about 5 minutes.
5. Add 10 µl of the ice-cold "2X buffer + ligase" solution. Immediately mix well, before the agarose hardens.
6. Incubate the ligation reaction at 16°C overnight.
7. The next day, proceed with part E or store your ligation reaction at 4°C until you're ready.

 

Transform the ligated DNA mixture into competent E. coli cells (next day)

Use sterile technique at all times when working with bacteria!

1. Transfer two tubes of competent E. coli cells (200 µl per tube) to your ice bucket. Allow >5 minutes to thaw if frozen.
2. Heat your ligation reaction (from Step D.7) at 70°C for about 10 minutes (to remelt the agarose).
3. Add 5 µl of the ligation reaction to the first tube of competent cells. Mix quickly by gentle shaking. To the second tube, add nothing; this is your negative control. Keep these tubes on ice for 30 minutes.
   Save the remainder of your ligation mix at 4°C for later use if needed.
4. After the 30 minutes on ice, transfer both tubes of transformed cells to a 42°C water bath. After exactly 90 seconds at 42°C, return the tubes to the ice bucket.
5. Chill both tubes on ice for 1 - 2 minutes; then sterilely add 0.8 ml SOC medium using a sterile 1 ml pipet.
6. Incubate the tubes of transformed cells at 37°C for 45 minutes.
   This step allows the bacteria to recover and express the antibiotic resistance gene.
7. Label two "SOB + ampicillin" agar plates. Sterilely transfer 200 µl of each transformation mixture to separate plates. Using a flame-sterilized bent glass rod, gently spread the cells over the surface of the agar.
   Save the remainder of the transformed cells at 4°C for later use if needed. To sterilize the bent glass rod, dip it into a plate full of ethanol, then briefly pass through a flame. When cooled, use the rod to spread the cells.
8. Once the liquid has been absorbed, incubate the plates, inverted, at 37°C overnight.
9. The next day, count the number of colonies on both plates. Store your plates in a 4°C refrigerator until next week.

• Overview
• Directions for Laboratory Period 1
• How to Interpret the Results of the Restriction Enzyme Analysis
• Directions for Laboratory Period 2
• The Alkaline Lysis Procedure for Preparing Plasmid DNA
• Directions for Laboratory Period 3

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