Restriction Enzyme Cloning

Restriction enzyme cloning is a bread-and-butter technique in molecular biology for modifying plasmids to contain genes or other DNA sequences of interest. While it may be more time consuming than some recently developed techniques, it is very reliable.

For background on restriction enzyme cloning and some pretty pictures, check out the Wikipedia page on this topic.

Materials

• Cloning plasmid (such as pUC19) and template for DNA fragment to be cloned
• Restriction Enzymes
• PCR Reagents
• DNA Ligase
• Electrocompetent cells or chemically competent cells of an appropriate cloning strain.
• Antibiotic Plates to your cloning plasmid and/or the antibiotic resistance gene you are cloning
• XGal and IPTG (for blue/white screens)

Step 1: Design Primers

Step 2: Perform PCR on template to amplify desired product with restriction sites

1. Using the primers you have designed to add restriction enzyme sites to the ends of your PCR fragment, perform PCR on your template to obtain your fragment.
2. Analyze your PCR product via agarose gel electrophoresis to ensure that you have obtained a product of the correct size. Use a gel extraction kit to perform a gel extraction of the desired band, or if the product is very pure, use a DNA purification kit to purify the DNA. Quantify DNA concentration.

Step 3: Restriction enzyme cleavage

1. Perform a restriction digest of both the cloning plasmid and the DNA fragment to be cloned to generate a linearized plasmid and DNA fragment with sticky ends. When considering how much DNA to add to the reaction, too much is preferable to too little. If performing a double digest (two restriction enzymes at the same time), be sure to use a buffer in which both enzymes have activity. Follow this protocol and the protocols that came with the restriction enzyme to plan your reaction.
2. Run the linearized plasmid and the digested fragment to be cloned (the whole reaction for both) on an agarose gel. Be sure to include uncut plasmid and uncut DNA fragment as controls in separate lanes so you can identify the "cut" version of each. Cut out your linearized plasmid and digested DNA fragment and purify them with a gel purification kit. Quantify DNA.

Optional Step 4: Phosphatase plasmid

If a high background of colonies of ligated vector (with no insert) is a problem (as may be the case when using only one restriction enzyme), you may use Calf Intestinal Phosphatase (CIP) to remove the phosphates from your linearized plasmid before proceeding to ligation. This will ensure that the linearized plasmid cannot ligate to itself, but must instead ligate to the insert to form a circular plasmid. You can likely ignore this step if you performed a double digest in the previous step.

Step 5: Ligate

Following the protocol for NEB T4 DNA Ligase, ligate your product into your linearize plasmid.

Step 6: Transform ligation reaction

1. Depending on the concentration of DNA used in the ligation reaction, transform 1-5 microliters of your ligation reaction into electrocompetent cells or chemically competent cells.
2. If transforming a selectable marker such as antibiotic resistance, plate the appropriate amount of transformed cells on plates containing the selective agent, grow overnight at 37 degrees (or the appropriate temperature for your plasmid, if other) and proceed to Step 7. If performing a blue/white screen or other kind of screen, proceed to Step 6A.

Step 6A: Blue/White Screening

If cloning a nonselectable piece of DNA, you will have to perform a screen of some kind to verify its presence. A common screen of this type is the blue/white screen. This is done with plasmids such as pUC19, where the multiple cloning site is located in the middle of the LacZ gene. As a result, a successful clone will disrupt the LacZ gene. Be sure to use an appropriate LacZ knockout strain when doing a blue/white screen.

1. Make plates to perform the blue/white screen by spreading X-Gal and IPTG on plates with the appropriate antibiotic for your plasmid. Stocks of X-Gal and IPTG should be available in the -20. If not, make your own!
2. Plate the appropriate amount of transformation on the plates and grow at 37 degrees overnight.
3. Plates the next day should have some blue, but mostly white colonies. Blue colonies indicate functional LacZ, which means that your insert was NOT cloned successfully. Therefore, you want to test white colonies, which indicate that LacZ has been disrupted. Pick several of these colonies and proceed to step 7

Step 7: Verify insert via PCR and sequencing

1. Perform colony PCR using a fraction of each colony on 5-10 colonies using primers outside the multiple cloning site of your vector. Use Taq polymerase for this. Run products on an agarose gel and check for product of expected size if insert is present.
2. Inoculate 5 mL of liquid LB media plus the appropriate antibiotic each with a colony with insert of correct size. Grow overnight
3. Isolate plasmid from overnight culture using a miniprep kit.
4. Sequence the insert using the same primers you used to verify insert size. Analyze sequence to confirm correct insert.