This is done with plasmids such as pUC19, where the multiple cloning site is located in the middle of the LacZ gene. A common screen of this type is the blue/white screen. If cloning a nonselectable piece of DNA, you will have to perform a screen of some kind to verify its presence. If performing a blue/white screen or other kind of screen, proceed to Step 6A. 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.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.You can likely ignore this step if you performed a double digest in the previous step.įollowing the protocol for NEB T4 DNA Ligase, ligate your product into your linearize plasmid. This will ensure that the linearized plasmid cannot ligate to itself, but must instead ligate to the insert to form a circular 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. Cut out your linearized plasmid and digested DNA fragment and purify them with a gel purification kit. Be sure to include uncut plasmid and uncut DNA fragment as controls in separate lanes so you can identify the "cut" version of each. Run the linearized plasmid and the digested fragment to be cloned (the whole reaction for both) on an agarose gel.Follow this protocol and the protocols that came with the restriction enzyme to plan your reaction. If performing a double digest (two restriction enzymes at the same time), be sure to use a buffer in which both enzymes have activity. When considering how much DNA to add to the reaction, too much is preferable to too little.
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.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. Analyze your PCR product via agarose gel electrophoresis to ensure that you have obtained a product of the correct size.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.Step 2: Perform PCR on template to amplify desired product with restriction sites Antibiotic Plates to your cloning plasmid and/or the antibiotic resistance gene you are cloning.Electrocompetent cells or chemically competent cells of an appropriate cloning strain.Cloning plasmid (such as pUC19) and template for DNA fragment to be cloned.Step 7: Verify insert via PCR and sequencing.Step 2: Perform PCR on template to amplify desired product with restriction sites.
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