Q8.   HOW SHOULD TEMPLATE DNA BE PREPARED FOR LA PCR?

               WHAT AMOUNT OF TEMPLATE DNA SHOULD BE USED?

The quality of template DNA is the key to LA PCR success. It is recommended to use the

purified DNA that is intact, free of nicks, and has undergone repeated extraction/purification.

DNA prepared simply with either heat treatment or protease digestion of cells without

further purification is not suitable for use as a template for the amplification of DNA

fragments longer than 10 kbp.

Refer to the Chapter 2 for standard protocols that are recommended for template

preparation from either human or E. coli genomic DNA.

Recommended amounts of template DNA to be used are as follows:

Human genomic DNA:          0.1-1 g/50 PCR reaction mix

E. coli genomic DNA:        10-100 ng/50  PCR reaction mix

lDNA or plasmid DNA:       0.5-2.5 ng/50  PCR reaction mix 

Effects of the quality of template DNA on LA PCR amplificatio

How does the quality of template DNA, including purification state and incurred

damage, affect LA PCR amplification?

As described below, DNA prepared by several methods were used as respective

templates in a 50 reaction mix for LA PCR amplification.

Template:
Human genomic DNA
Amplified region:

-globin cluster

Amplified sizes:

17.5 kbp