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DNA Cloning by PCR (CAT#: STEM-MB-0232-WXH)

Introduction

In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest. PCR cloning differs from traditional cloning in that the DNA fragment of interest, and even the vector, can be amplified by the Polymerase Chain Reaction (PCR) and ligated together, without the use of restriction enzymes. <br />PCR based cloning is incredibly versatile and allows for nearly any piece of DNA to be placed into a backbone vector of choice with minimal limitations. PCR cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate. It allows for the cloning of DNA fragments that are not available in large amounts.




Applications

Cloning of DNA fragments that are not available in large amounts.

Procedure

1.Run PCR and purify the PCR product
2.Digest your DNA
3.Isolate your insert and vector by gel purification
4.Ligate your insert into your vector
5.Transformation
6.Isolate the Finished Plasmid
7.Verify your Plasmid by Sequencing

Materials

The basic PCR primers for molecular cloning consist of:
Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction enzyme digestion
Restriction Site: Your chosen restriction site for cloning
Hybridization Sequence: The region of the primer that binds to the sequence to be amplified

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