THE CLONING OF
PLASMID AND SPINACH DNA

Teri Curtis
Adapted From Labs Written By Dr. Karen Armstrong-Malatesta and Judy Brown 1993 Woodrow Wilson Biology Institute

INTRODUCTION

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:

STUDENT/CLASS TIME REQUIRED

MATERIALS:

1. DNA EXTRACTION FROM SPINACH:

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.

2. RESTRICTION ENZYME DIGESTION:

1. pUC18 30ug/100 ul from Modern Biology, Inc # 3-14-30 at $ 26.25
   or
   pUC8 10ug/100 ul 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 ul range)
   (Modern Biology, Inc carries an inexpensive model)

   Distilled water

   Genomic DNA extracted from spinach

2b.

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 100ug/400ul DNA Standard II from Modern Biology at $36.75 (enough for 30-40 lanes).

3. Ligation:

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:

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

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 65deg.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:

   TUBE A: Plasma digest
   10X RE buffer = 2ul
   plasmid DNA = 3ul
   Spinach DNA = 0ul
   RE HindIII = 1ul
   H20 = 14ul

   TUBE B: Uncut plasmid
   10X buffer = 2ul
   plasmid DNA = 3ul
   Spinach DNA = 0ul
   RE HindIII = 0ul
   H20 = 15ul

   TUBE C: Spinach DNA digest
   10X buffer = 5ul
   plasmid DNA = 0ul
   spinach DNA = 30ul
   RE HindIII = 5ul
   H20 = 10ul

   TUBE D: Uncut Spinach DNA
   10X buffer = 5ul
   plasmid DNA = 0ul
   spinach DNA = 30ul
   RE HindIII = 0ul
   H20 = 15ul

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

3. When the incubation time is complete, heat inactivate the RE's by putting your digests in the 65deg.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 microl of loading dye to each tube. Then add the following (remember to change the tips of your micropipettor in between different reagents):
   tube #1 5ul digested plasmid DNA
   tube #2 5ul undigested plasmid DNA
   tube #3 5ul digested spinach DNA
   tube #4 5ul 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 ul from tube 1
   Lane 2 - " " tube 2
   Lane 3 - " " tube 3
   Lane 4 - " " tube 4
   Lane 5 - 15 ul 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:

   25 ul 2X buffer
   10 ul plasmid digest
   10 ul spinach DNA digest
   4 ul T4 DNA ligase
   24 ul dH2O

2. 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 ul                     100 ul
(nutrient agar)

LB/amp+XGal                 100 ul                     100 ul
(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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