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Cloning of Plasmid and Spinach DNA

Author: Teri Curtis
Woodrow Wilson Biology Institute
1993


This work is adapted From Labs Written By Dr. Karen Armstrong-Malatesta and Judy Brown

Teacher Pages

Introduction: The purpose of this lab is to ligate fragments of genomic DNA from spinach into a vector plasmid; this recombinant DNA is then used to transform Escherichia coli cells.

The major steps in this lab are as follows:

  1. Students will first extract genomic DNA from spinach.

  2. Students will perform a restriction enzyme digestion on both the spinach DNA and the plasmid using the same restriction enzyme.

    Optional: If electrophoresis equipment is available, students may run a gel on a portion of each digest (the spinach and the plasmid) to check for the presence of cut DNA in each digest (see pages 247-275 in DNA Science).

  3. Students will ligate the cut plasmid DNA with the cut spinach DNA in order to create a recombinant plasmid.

  4. Students will transform E. coli cells with their recombinant DNA molecule.

Target Group: Second year biology students.

Student/class time required
(in periods; 1 period = 50 min.):

  • prelab--1
  • spinach extraction--1
  • restriction enzyme digest--1
  • ligation--1
  • transformation--1 or 2
  • analysis--1
  • Materials (The following list provides
    enough material for 20 students working in pairs:

    1. DNA extraction from spinach:

  • 300 g of spinach
  • 1 liter - 20% liquid detergent (200 ml of Palmolive or Dawn liquid detergent diluted to a total volume of 1L).
  • Hot water bath at 65 degree C
  • Cheesecloth - approx. 2 yd.2
  • 100 ml - 15% meat tenderizer (15g of freshly opened Adolf's dissolved in enough water to make 100 ml)
  • 100 ml 95% ethanol - ice cold
  • 250 ml NaCl solution - Dissolve 29.2 g of NaCl (non-iodinized) in enough distilled water to make a final volume of 250 ml
  • 20 -30 ml centrifuge tubes
  • 10 - mortar and pestles
  • 10 - 5 to 10 ml centrifuge tubes (for storing DNA)
  • 10 ml pipettes and bulbs
  • 10 Pasteur pipettes (flame the tip of each Pasteur pipette and it into a small hook for spooling DNA)
  • 10 to 20 small beakers stirring rods, sand
  • Note: If students have difficulty in obtaining adequate spinach DNA using the method in this lab, have them try the method in "Separation of DNA from Onion" by Ellen Avery, also found in this module.

    2a. Restriction enzyme digestion:

    plasmid DNA, order either:
  • pUC18 30microg/100 microl from Modern Biology, Inc
    # 3-14-30 at $ 26.25
    or
  • pUC8 10microg/100 microl from Carolina Biological
    # 21-146 at $49.95
  • or have students perform a plasmid miniprep on an E. coli strain containing the plasmid following the miniprep directions outlined in the labs by Peggy Campbell in this module or in DNA Science pages 313-323.

    HindIII restriction enzyme, order either:

  • 1000 units from Modern Biology, Inc
    #3-9A-1 at $21.00
    or
  • 2000 units from Carolina Biological
    #21-1690 at $26.95

    (both sent with restriction enzyme specific buffer)

  • microcentrifuge tubes (1.5 ml size)
  • 10 micropipettor + tips (1- 20 microl range) (Modern Biology, Inc carries an inexpensive model)
  • Distilled water
  • Genomic DNA extracted from spinach
  • 2b.

    For agarose gel electrophoresis see DNA Science pages 256-270 for general instructions for running a gel. Modern Biology, Inc has a Reagent Package for DNA Gel which includes agarose, electrophoresis buffer, loading buffer (dye), and stain (either methylene blue or ethidium bromide):

  • order #3-1 for pkg. w/methylene blue for $51.45
  • order #3-2 for pkg. w/ethidium bromide for $51.45
  • For 1kb marker order # 3-12 100microg/400microl DNA Standard II from Modern Biology at $36.75 (enough for 30-40 lanes).
  • 3. Ligation:

  • 50 microl of T4 DNA ligase (2X ligase buffer included) from Carolina #21-1740 at $53.80

    Note: The ligase must be kept on ice until the moment it is needed for the ligation reactions.

  • each team should already have: plasmid digest and genomic DNA (spinach) digest
  • 4. Rapid Colony Transformation:

    For complete list of materials see "Teacher Preparation: Part I. Rapid Colony Transformation" by Peggy Campbell.

  • Replace the LB agar+amp with LB agar+amp+XGal or Nutrient agar+amp+Xgal.

  • From Modern Biology, 400 ml of Nutrient agar+amp+XGal is #4-4X at $26.25, and 400 ml of Nutrient agar is #4-3 at $13.15.

  • E. coli strain, order either:

  • plate of strain DH5 from Modern Biology #4-5 at $7.35.
    or
  • slant of strain JM101 from Carolina Biological #21-1561 at $6.95.
  • Student Instructions

    Introduction: In this lab you will have the opportunity to make a recombinant DNA molecule by combining a fragment of spinach DNA with a vector plasmid.

    First, you will extract DNA from spinach. Second, you will cut this spinach DNA with the restriction enzyme(RE) HINDIII. You will also cut the vector plasmid (pUC18, pUC8 or some other appropriate plasmid provided by your instructor) with HINDIII (It's critical that the plasmid DNA and the genomic DNA from the spinach are cut with the same restriction enzyme. Why?).

    You will then mix the digested spinach DNA and the digested plasmid together. With skill and a little luck, the sticky ends of the plasmid will meet the sticky ends of the spinach DNA fragments (which you created with the RE HINDIII) to form a recombinant plasmid. Ligase will be added to catalyze the formation of covalent bonds between the fragments of this DNA molecule.

    Next, you will use your newly ligated DNA molecule to transform E.coli cells. How will you know if your transformation is successful?

    The plasmid which you are using in this lab has been genetically engineered to exhibit certain traits. The plasmid has a gne for ampicillin resistance. It also carries a portion of the coding regions for the E.coli LacZ gene. This segment of the LacZ gene is known as the alpha-complementary region.

    In its entirety, the LacZ gene encodes the enzyme beta-galactosidase; this enzyme metabolizes lactose. In certain strains of E coli which encode the rest of the of the LacZ gene and also carry a plasmid with an alpha-complementary region, the colonies are normally blue in color on plates containing a chromogenic substrate (XGal) of beta-galactosidase.

    Beta-galactosidase will only be produced if the entire LacZ gene is present. The plasmid in this lab has been engineered so that its restriction enzyme sites are located in the LacZ-encoding region. Therefore, when a fragment of DNA is inserted into this plasmid, the LacZ (alpha-complementary) region is disrupted. The result is that the LacZ gene no longer works and the E.coli carrying this recombinant plasmid are white instead of blue.

    Procedure

    Part I. Spinach Extraction

    1. Add 12 g of fresh green spinach leaves, 1 ml of water and a few grains of sand to a mortar and pestle. Grind the leaves until they look creamed.

    2. Add 1 ml of 20% detergent and 9 g of salt to the mush and mix (this step helps lyse the cell walls and membranes).

    3. Pour or scrape the spinach mush into a large tube and incubate in a 65 degree C hot water bath for 10 minutes.

    4. Place mush on ice to stop the process. Filter the mush through 4 layers of cheesecloth into a beaker. Your filtrate can be stored overnight in the refrigerator at this point.

    5. Pour 6 ml of the filtrate into a centrifuge tube and add 1 ml of 15% meat tenderizer (Adolf's) solution. Gently invert the tube to mix (this step helps remove protein from the DNA).

    6. Slowly pour 6 ml of cold 95% ethanol down the side of the tube. Spool the DNA onto hooked Pasteur pipette by gently stirring at the interface of the alcohol and filtrate. If you are unable to get the DNA to spool, try centrifuging the mixture. The DNA looks gelatinous (like clear mucus).

    7. Carefully put the rod containing the DNA into a test tube (5 to 10 ml) containing 3ml of the salt solution.

    Part II. Restriction Enzyme Digestion

    1. Set-up the following digests in microcentrifuge tubes adding reagents in the order listed (be sure to label your tubes A, B, C, D). Add reagents to the tubes in the order that they are listed from left to right:
                             10X RE           plasmid          Spinach         RE
      Tube                   buffer             DNA             DNA          HindIII          
      H2O
      
      A. Plasmid digest     2 microl         3 microl          0 micorl      1microl        
      14 microl
             
      B. Uncut plasmid      2 microl         3 microl          0  microl     0 microl     
      15 microl
      
      C. Spinach DNA 
      digest                5 microl         0 microl          30 microl     5 microl       
      10 microl
      
      D. Uncut Spinach
      DNA                   5 microl         0 microl          30 microl     0 micro        
      15 microl
      

    2. Incubate these digests in a 37 degree C water bath for 30 min.

    3. When the incubation time is complete, heat inactivate the RE's by putting your digests in the 65 degree C water bath for 5 min. Your digests may be stored in the refrigerator overnight.

    Optional: If you have electrophoresis equipment, it's a good idea to make sure that you actually have digested plasmid DNA (tube A), undigested plasmid (tube B), digested spinach DNA (tube C),and undigested spinach DNA (tube D). If equipment is not available, you should skip down to Part III. Ligation.

    Otherwise, follow instructions from your teacher for setting up your agarose gel and electrophoresis equipment. After you have poured your gel, do the following:

    Number fresh microtubes 1 to 4. Add 4 micol of loading dye to each tube. Then add the following (remember to change the tips of your micropipettor in between different reagents):

    tube #1 5microl digested plasmid DNA
    tube #2 5microl undigested plasmid DNA
    tube #3 5microl digested spinach DNA
    tube #4 5microl undigested spinach DNA

    Tap each tube to insure that the drops of loading dye and DNA are brought together.

    Load the mixtures from your tubes into your gel in the following manner:

    Lane 1 - 9 microl from tube 1
    Lane 2 - " " " tube 2
    Lane 3 - " " " tube 3
    Lane 4 - " " " tube 4 Lane 5 - 15 microl of DNA Standard II (dye included)

    Follow teacher instructions for running the gel.

    Part III. Ligation

    1. Place the following substances in the order listed into a microfuge tube:

    2. 25 microl 2X buffer
    3. 10 microl plasmid digest
    4. 10 microl spinach DNA digest
    5. 4 microl T4 DNA ligase
    6. 24 microl dH2O
    7. Incubate at room temperature for 2 to 3 hours (or overnight in the refrigerator).

    Part IV. Transformation of E.coli with ligated DNA

    1. After the ligation mixes have incubated, you are ready to perform the transformation of E.coli cells. Follow the Rapid Colony Transformation method as described by Peggy Campbell in this module or on pages 303-307 of DNA Science with the following modifications:

      A. Each place that the lab procedure refers to +pAMP or -pAMP, you substitute +pUC18(or pUC8) and -pUC18 (or pUC8).

      B. You also will use two LB plates (no ampicillin) or two nutrient agar plates (no ampicillin). However, instead of using two LB/amp plates you will use either two LB+amp+XGal plates or two nutrient agar+amp+XGal plates.

      C. You should refer to the following matrix when plating out your E. coli cells:

                         +pUC18           -pUC18
    Agar used          (or pUC8)        (or pUC8) 
    
    LB                 100 microl       100 microl
    (nutrient agar)
    
    LB/amp+XGal        100 microl       100 microl
    (nutrient agar+ 
    amp+XGal)
    

    Part V. Analysis

    1. Observe the colonies through the bottom of the culture. Record your results. Are your results what you expected?

    2. What does the presence or absence of colonies on each plate indicate?

    3. Did transformation take place? What evidence do you have to support your conclusion? Explain.

    4. Were you successful in transforming E.coli cells with a recombinant plasmid? How do you know?


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