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How do you choose restriction enzymes for a plasmid?

How do you choose restriction enzymes for a plasmid?

When selecting restriction enzymes, you want to choose enzymes that:

  1. Flank your insert, but do not cut within your insert.
  2. Are in the desired location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid.

What are considerations for working with restriction enzymes in a restriction digest?

To ensure efficient activity of restriction endonucleases, they should be properly stored and used before their expiration date per supplier recommendations. In general, enzymes should be kept at –20⁰C in a non–frost-free freezer, preferably in small aliquots, to maintain activity and to minimize freeze-thaw cycles.

How many enzymes should be used in a plasmid DNA and an insert if there is a desire to ligate the insert in the vector to a specific orientation?

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Usually, scientists select two different enzymes for adding an insert into a vector (one enzyme on the 5′ end and a different enzyme on the 3′ end). This ensures that the insert will be added in the correct orientation and prevents the vector from ligating to itself during the ligation process.

How can restriction enzymes be used to create a recombinant plasmid?

The basic steps are:

  1. Cut open the plasmid and “paste” in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).
  2. Insert the plasmid into bacteria.
  3. Grow up lots of plasmid-carrying bacteria and use them as “factories” to make the protein.

What would be the best choice of restriction enzyme’s to use for this purpose?

Your best choice would be a restriction enzyme within the multiple cloning sequence (MCS) e.g. EcoRI and HindIII. MCS is within the lacz gene. This means you can use blue-white selection in E. coli.

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What factors affect restriction enzyme digestion?

The digestion activity of restriction enzymes depends on the following factors:

  • Temperature: Most endonucleases digest the target DNA at 37 °C with few exceptions.
  • Cofactors: Restriction endonucleases require certain cofactors or combination of cofactors to digest at the recognition site.

What is a restriction enzyme do?

A restriction enzyme is an enzyme isolated from bacteria that cuts DNA molecules at specific sequences. The isolation of these enzymes was critical to the development of recombinant DNA (rDNA) technology and genetic engineering.

What are restriction enzymes examples?

SmaI is an example of a restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end. Other restriction enzymes, like EcoRI, cut through the DNA strands at nucleotides that are not exactly opposite each other.