Using PCR to determine orientation of plasmid insert

[CPScholar]

Could someone please explain to me how PCR can be used to determine the orientation of an insert? I’ve been trying to understand this process but I am having a hard time.

[oppox]

Where the insert is located depends on where u cut the vector, Im not familiar with a way to locate it if u dont know where its located.
But u can find out if it is your insert and if its turned in the right direction by using a primer that fits the vector closly to where u think the gene is and a primer that fits your gene.

[scthawke]

Yeah I agree basically with CPScholar.

You use a PCR primer that binds in the vector and close to the site you put in your insert in. YOu can often find sites for m13 forward or reverse sites in many vectors, so these PCR primers are often found in many labs. Then you need another PCr primer in the insert itself (it is best if it is towards one end of the insert). This is the hard one to get and often you have to get this primer made.

Now in one orientation this primer will work with your primer in the vector and produce a PCr product of a known size. If your insert is in the other direction then the PCR primer in the insert will bind such that it points away from the one in your vector and you will not got a PCr product of any size.

Often people will setup two PCRs, geared up to work such that if the insert is in one oritentation then PCR A will work, while if it is in the other orientation then PCR B will work.

Simple huh?

In general however determining the orientation of an insert in this manner is not used. Restriction enzymes are generally an easier option.

[CPScholar]

Thanks! Both replys were definitely helpful.

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